Real-time pcr for the detection of pathogens

ABSTRACT

Disclosed herein are methods for detecting presence of one or more of  Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma  spp.,  Bartonella  spp.,  Streptococcus agalactiae , and  Neisseria meningitidis  nucleic acids in a sample, such as a biological sample obtained from a subject, or an environmental sample. This disclosure also provides probes, primers, and kits for detecting one or more of  Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma  spp.,  Bartonella  spp.,  Streptococcus agalactiae , and  Neisseria meningitidis  in a sample.

CROSS REFERENCE TO RELATED APPLICATION

This claims the benefit of U.S. Provisional Application No. 61/642,091,filed May 3, 2012, which is incorporated herein by reference in itsentirety.

FIELD

This disclosure concerns methods and compositions related to thedetection of pathogens, particularly utilizing real-time PCR.

BACKGROUND

Many pathogens have major public health and economic impact. Pathogensmay be spread in the community or in a clinic or hospital setting, andmultidrug resistance is a growing problem in many pathogens.Furthermore, although a presumptive clinical diagnosis can often be madethrough symptomology, a laboratory identification determining theetiology of a disease is critical to establish the correct course oftreatment. Current tests for many pathogens are neither highly sensitivenor specific, and in some cases require an acute and convalescentpatient serum (paired serum) for clear identification. Thus, a needremains for rapid, cost-effective, sensitive, and specific assays formany pathogens. In particular, there is a need for assays for diagnosingand differentiating major pathogens of childhood and neonatal infection,which cause significant neonatal mortality throughout the world.

SUMMARY

Disclosed herein are methods for detecting presence of one or more ofAcinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae (Group B Streptococcus; GBS), and/orNeisseria meningitidis nucleic acids in a sample, such as a biologicalsample obtained from a subject, or an environmental sample. Inparticular, these pathogens are some of the most common causes ofinfection in neonates and young children, and are major causes ofmortality in children under the age of five. The methods provided hereininclude simultaneous multipathogen detection assays, which can be usedto diagnose and differentiate causes of neonatal infection. In addition,since all of these assays target infectious agents, all populationgroups may benefit from the advancements described herein. Depending onthe specific circumstances and epidemiological data associated withsporadic and/or outbreak-linked cases, the disclosed methods can providea valuable diagnostic tool for the clinician and medical epidemiologistcharged with determining the etiology of a disease.

The disclosed methods can be used to detect presence of one or more (orany combination of two or more thereof) of Acinetobacter baumannii,Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii,Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcusaureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasmaurealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp.,Streptococcus agalactiae, and/or Neisseria meningitidis nucleic acids ina sample, for example, by contacting a sample with one or more of theprobes disclosed herein (such as one or more of SEQ ID NOs: 14, 17, 20,23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 56, 60, and 64) and detectinghybridization of one or more of the probes with a nucleic acid in thesample. The disclosed methods provide rapid, sensitive, and specificdetection of these organisms, for example, utilizing real-timesimultaneous multipathogen detection or a multiplex real-time PCR assay.

In some embodiments, the disclosed methods further include amplifyingone or more (or any combination of two or more thereof) of anAcinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, and/or Neisseria meningitidis nucleicacid, for example utilizing one or more primers (such as one or more ofSEQ ID NOs: 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 31, 33,34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 54, 55, 58, 59, 62,and/or 63).

This disclosure also provides kits for detecting one or more ofAcinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, and/or Neisseria meningitidis in asample, for example, including one or more of the probes and primersdisclosed herein.

The foregoing and other features, of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C is a series of graphs showing the effect of lytic enzymetreatment on extraction of nucleic acid from blood specimens. Average Ctvalue of individual real-time PCR reactions (n=4) containing TNAextracted from healthy donor blood spiked with serial dilutions of S.aureus (FIG. 1A) or K. pneumoniae (FIG. 1B) without treatment, afterincubation with TE buffer alone, or after treatment with TE buffer withlytic enzymes (lysozyme, lysostaphin, and mutanolysin) at 37° C. for 30minutes. FIG. 1C shows Ct values of serial dilutions of K. pneumoniaespiked into saline (to mimic NP/OP swab) or blood. Error bars displaystandard deviation. * p<0.0001 compared to no treatment. ^(#) p<0.05compared to same concentration of organisms in saline.

FIG. 2A-B is a pair of graphs showing concordance between replicates ofprimary clinical specimens tested on a TAQMAN® array card (TAC).Concordance between replicate results for nasopharyngeal/oropharyngeal(NP/OP) (FIG. 2A) and blood (FIG. 2B) specimens tested using TAC. Datashown are total number of specimens identified as positive in at leastone replicate reaction (white bars) and proportion of positive specimensfor which greater than 50% of replicates were positive (shaded bars).Number of replicates tested varied by target and specimen type; alltargets were tested in ≧2 replicates. Total number of specimens tested,NP/OP (n=124), blood (n=661). ADEV, Adenovirus; BOP1, Bordetellapertussis; ENTV, Enterovirus; FLUA, Influenza A; GBST, Group BStreptococcus; HMPV, Human Metapneumovirus; RESV, Respiratory SyncytialVirus; RHIV, Rhinovirus; STPN, Streptococcus pneumoniae; URUP,Ureaplasma spp.; CYMV, Cytomegalovirus; ECSH, Escherichia coli/Shigellaspp.; GAST, Group A Streptococcus; HIAT; Haemophilus influenzae; KLPN,Klebsiella pneumoniae, PSAE, Pseudomonas aeruginosa, SALS, Salmonellaspp.; STAU, Staphylococcus aureus.

FIGS. 3A and B are a pair of diagrams showing effect of enzyme system ondetection of pathogen targets in primary clinical specimens. Data shownare difference in Ct value between reactions using Quanta One-stepRT-PCR TOUGHMIX® and AGPATH-ID™ One-step RT-PCR kit when testing TNAextracted from NP/OP swabs (FIG. 3A) or blood (FIG. 3B). Each data pointrepresents the difference in Ct value between the two reactions for anindividual clinical specimen. Median difference is indicated (−) forassays with ≧2 positive results. *Targets that were only detected usingAGPATH-ID™ always occurred when Ct values were >33.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases, and three letter code for aminoacids, as defined in 37 C.F.R. §1.822. In at least some cases, only onestrand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand.

SEQ ID NO: 1 is an exemplary Acinetobacter baumannii oxa-51 nucleic acidsequence.

SEQ ID NO: 2 is an exemplary Chlamydia trachomatis tmRNA nucleic acidsequence.

SEQ ID NO: 3 is an exemplary Escherichia coli uidA nucleic acidsequence.

SEQ ID NO: 4 is an exemplary Klebsiella pneumoniae nifA nucleic acidsequence.

SEQ ID NO: 5 is an exemplary Moraxella catarrhalis purH nucleic acidsequence.

SEQ ID NO: 6 is an exemplary Pneumocystis jirovecii dhps nucleic acidsequence.

SEQ ID NO: 7 is an exemplary Pseudomonas aeruginosa gyrB nucleic acidsequence.

SEQ ID NO: 8 is an exemplary Staphylococcus aureus gsf nucleic acidsequence.

SEQ ID NO: 9 is an exemplary Toxoplasma gondii ssrRNA nucleic acidsequence.

SEQ ID NO: 10 is an exemplary Ureaplasma parvum ure nucleic acidsequence.

SEQ ID NO: 11 is an exemplary Ureaplasma urealyticum mba nucleic acidsequence.

SEQ ID NOs: 12-14 are exemplary Acinetobacter baumannii oxa-51 primerand probe nucleic acid sequences.

SEQ ID NOs: 15-17 are exemplary Pseudomonas aeruginosa gyrB primer andprobe nucleic acid sequences.

SEQ ID NOs: 18-20 are exemplary Klebsiella pneumoniae nifA primer andprobe nucleic acid sequences.

SEQ ID NOs: 21-23 are exemplary Toxoplasma gondii ssrRNA primer andprobe nucleic acid sequences.

SEQ ID NOs: 24-26 are exemplary Moraxella catarrhalis purH primer andprobe nucleic acid sequences.

SEQ ID NOs: 27-29 are exemplary Escherichia coli/Shigella spp. primerand probe nucleic acid sequences.

SEQ ID NOs: 30-32 are exemplary Staphylococcus aureus gsf primer andprobe nucleic acid sequences.

SEQ ID NOs: 33-35 are exemplary Pneumocystis jirovecii dhps primer andprobe nucleic acid sequences.

SEQ ID NOs: 36-38 are exemplary Chlamydia trachomatis tmRNA primer andprobe nucleic acid sequences.

SEQ ID NOs: 39-41 are exemplary Ureaplasma urealyticum mba primer andprobe nucleic acid sequences.

SEQ ID NOs: 42-44 are exemplary Ureaplasma parvum ure primer and probenucleic acid sequences.

SEQ ID NOs: 45-47 are exemplary Ureaplasma spp. ure primer and probenucleic acid sequences.

SEQ ID NOs: 48-51 are exemplary Bartonella spp. ssrA primer and probenucleic acid sequences.

SEQ ID NO: 52 is an exemplary Bartonella ssrA nucleic acid sequence.

SEQ ID NO: 53 is an exemplary Group B Streptococcus cfb nucleic acidsequence.

SEQ ID NOs: 54-56 are exemplary Group Streptococcus cfb primer and probenucleic acid sequences.

SEQ ID NO: 57 is an exemplary Klebsiella pneumoniae diguanylate cyclasenucleic acid sequence.

SEQ ID NOs: 58-60 are exemplary Klebsiella pneumoniae diguanylatecyclase primer and probe nucleic acid sequences.

SEQ ID NO: 61 is an exemplary Neisseria meningitidis sodC nucleic acidsequence.

SEQ ID NOs: 62-64 are exemplary Neisseria meningitidis sodC primer andprobe nucleic acid sequences.

SEQ ID NOs: 65-67 are exemplary Salmonella spp. ttrRsBCA primer andprobe nucleic acid sequences.

DETAILED DESCRIPTION I. Abbreviations

BAL: bronchoalveolar lavage

GBS: Group B Streptococcus

NP: nasopharyngeal

NTC: no template control

OP: oropharyngeal

TAC: TAQMAN array card

TNA: total nucleic acids

UTM: universal transport medium

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. As used herein,“comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements. Itis further to be understood that all base sizes or amino acid sizes, andall molecular weight or molecular mass values, given for nucleic acidsor polypeptides are approximate, and are provided for description. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety for allpurposes.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. In case of conflict,the present specification, including explanations of terms, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Acinetobacter baumannii: An aerobic gram-negative bacterium which cancause pneumonia, urinary tract infection, and necrotizing fasciitis.Many strains of A. baumannii are antibiotic resistant and it is anincreasingly common nosocomial infection, for example, in intensive careunits (such as neonatal intensive care units). A. baumannii can alsocolonize solutions (such as irrigating or intravenous solutions).Nucleic acid and protein sequences for A. baumannii are publiclyavailable. For example, GenBank Accession Nos. NC_(—)011586,NC_(—)011595, NC_(—)010611, NC_(—)009085, and NC_(—)010410 provideexemplary A. baumannii genome sequences, all of which are incorporatedby reference as provided by GenBank on Apr. 30, 2012.

The A. baumannii oxa-51 gene encodes a beta-lactamase. Exemplary A.baumannii oxa-51 nucleic acid sequences include GenBank Accession Nos.AJ309734 and DQ385606, both of which are incorporated by reference aspresent in GenBank on Apr. 30, 2012. An exemplary Acinetobacterbaumannii nucleotide sequence of oxa-51 is found at GenBank AccessionNo. AJ309734 (SEQ ID NO: 1).

Bartonella spp.: A genus of gram-negative bacteria that infect a widevariety of mammalian hosts, including humans. Bartonella are transmittedby blood-sucking insects (for example, ticks, fleas, and lice). Highprevalence of Bartonella bacteremia has been reported in populations ofrodents, cats, and ruminants worldwide. There are at least 30 knownspecies and subspecies of Bartonella. Nucleic acid and protein sequencesfor Bartonella spp. are publicly available. For example, GenBankAccession Nos. NC_(—)005955 (B. quintana), NC_(—)005956 (B. henselae),NC_(—)010161 (B. tribocorum), NC_(—)012846 (B. grahamii), andNC_(—)008783 (B. bacilliformis) provide exemplary Bartonella genomesequences, all of which are incorporated by reference as provided byGenBank on Apr. 30, 2012. One of ordinary skill in the art can identifyadditional Bartonella spp., for example utilizing the NCBI TaxonomyBrowser (e.g., www.ncbi.nlm.nih.gov/Taxonomy/).

The Bartonella ssrA RNA (also known as transfer-messenger RNA; tmRNA) isa single-copy prokaryotic-specific molecule involved in processing ofincomplete peptides and resolution of stalled ribosomes duringtranslation. Exemplary Bartonella ssrA nucleic acid sequences includeGenBank Accession Nos. JNO29766, BX897700 (1020848 . . . 1021176),JNO29785, BX87699 (1215947 . . . 1216279), JNO29796, AM260525 (1675346 .. . 1675682), JNO29795, NC_(—)012846.1 (1542284 . . . 1542620),JNO29794, and NC_(—)008783.1 (955069 . . . 955373), all of which areincorporated herein by reference as provided by GenBank on Apr. 30,2012. An exemplary Bartonella nucleotide sequence of ssrA is found atGenBank Accession No. NC_(—)005955 (1020727-1021342) (SEQ ID NO: 52).

Chlamydia trachomatis: A gram-negative bacterium that is a commonsexually transmitted disease. C. trachomatis can also be transmittedfrom an infected mother, resulting in a potentially life-threateningrespiratory infection in neonates. Nucleic acid and protein sequencesfor C. trachomatis are publicly available. For example, GenBankAccession Nos. NC_(—)012687, NC_(—)000117, NC_(—)007429, NC_(—)010280,and NC_(—)015744 provide exemplary C. trachomatis genome sequences, allof which are incorporated by reference as provided by GenBank on Apr.30, 2012.

The C. trachomatis tmRNA is an RNA with both tRNA and mRNAcharacteristics. Exemplary C. trachomatis tmRNA nucleic acid sequencesinclude GenBank Accession Nos. NC_(—)000117 (20663-21082) andNC_(—)007429 (21258-21677; complement), both of which are incorporatedherein by reference as present in GenBank on Apr. 30, 2012. An exemplaryChlamydia trachomatis nucleotide sequence of tmRNA is found at GenBankAccession No. NC_(—) 000117 (20663-21082) (SEQ ID NO: 2).

Escherichia coli: A Gram-negative, rod-shaped bacterium that is commonlyfound in the lower intestine of warm-blooded organisms, where it (andrelated bacteria) constitute about 0.1% of gut flora. Most E. colistrains are harmless, but some serotypes (for example 0157:H7 and0104:H4) can cause serious disease in humans. Pathogenic E. coli isfrequently contracted via contaminated food or water. The harmlessstrains are part of the normal flora of the gut, and can benefit theirhosts by producing vitamin K₂ and by preventing the establishment ofpathogenic bacteria within the intestine.

Nucleic acid and protein sequences for E. coli are publicly available.For example, GenBank Accession Nos. NC_(—)011751, NC_(—)011742,NC_(—)011415, NC_(—)011601, and NC_(—)011353 provide exemplary E. coligenome sequences, all of which are incorporated by reference as providedby GenBank on Apr. 30, 2012.

The E. coli uidA gene encodes a β-D-glucuronidase. Exemplary E. coliuidA nucleic acid sequences include GenBank Accession Nos. NC_(—)011601(1769353-1771164, complement), NC_(—)000913 (1692284-1694095), andNC_(—)011751 (192037-1930848, complement), all of which are incorporatedherein by reference as present in GenBank on Apr. 30, 2012. An exemplaryE. coli nucleotide sequence of uidA is found at GenBank Accession No.NC_(—)000913 (1692284-1694095) (SEQ ID NO: 3).

Fluorophore: A chemical compound, which when excited by exposure to aparticular stimulus, such as a defined wavelength of light, emits light(fluoresces), for example at a different wavelength (such as a longerwavelength of light).

Fluorophores are part of the larger class of luminescent compounds.Luminescent compounds include chemiluminescent molecules, which do notrequire a particular wavelength of light to luminesce, but rather use achemical source of energy. Therefore, the use of chemiluminescentmolecules (such as aequorin) eliminates the need for an external sourceof electromagnetic radiation, such as a laser.

Examples of particular fluorophores that can be used in the probes andprimers disclosed herein are known to those of ordinary skill in the artand include those provided in U.S. Pat. No. 5,866,366 to Nazarenko etal., such as 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid;acridine and derivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide; BrilliantYellow; coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), QFITC (XRITC), 6-carboxy-fluorescein(HEX), and TET (tetramethyl fluorescein); fluorescamine; IR144; IR1446;Malachite Green isothiocyanate; 4-methylumbelliferone;ortho-cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate, and succinimidyl 1-pyrene butyrate; ReactiveRed 4 (CIBACRON™ Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate,N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC); sulforhodamine B;sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine101 (Texas Red); riboflavin; rosolic acid and terbium chelatederivatives; LightCycler Red 640; Cy5.5; and Cy56-carboxyfluorescein;boron dipyrromethene difluoride (BODIPY); acridine; stilbene; Cy3; Cy5,VIC® (Applied Biosystems); LC Red 640; LC Red 705; and Yakima yellowamongst others. Additional examples of fluorophores include Quasar® 670,Quasar® 570, CAL Fluor® Red 590, CAL Fluor® Red 610, CAL Fluor® 615, CALFluor® Red 635, CAL Fluor® Green 520, CAL Fluor® Gold 540, and CALFluor® Orange 560 (Biosearch Technologies, Novato, Calif.).

Other suitable fluorophores include those known to those of ordinaryskill in the art, for example those available from Molecular Probes/LifeTechnologies (Carlsbad, Calif.). In particular examples, a fluorophoreis used as a donor fluorophore or as an acceptor fluorophore.

“Acceptor fluorophores” are fluorophores which absorb energy from adonor fluorophore, for example in the range of about 400 to 900 nm (suchas in the range of about 500 to 800 nm). Acceptor fluorophores generallyabsorb light at a wavelength which is usually at least 10 nm higher(such as at least 20 nm higher) than the maximum absorbance wavelengthof the donor fluorophore, and have a fluorescence emission maximum at awavelength ranging from about 400 to 900 nm. Acceptor fluorophores havean excitation spectrum that overlaps with the emission of the donorfluorophore, such that energy emitted by the donor can excite theacceptor. Ideally, an acceptor fluorophore is capable of being attachedto a nucleic acid molecule.

In a particular example, an acceptor fluorophore is a dark quencher,such as Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular Probes), BLACKHOLE QUENCHERS™ (Biosearch Technologies; such as BHQ0, BHQ1, BHQ2, andBHQ3), ECLIPSE™ Dark Quencher (Epoch Biosciences), or IOWA BLACK™(Integrated DNA Technologies). A quencher can reduce or quench theemission of a donor fluorophore. In such an example, instead ofdetecting an increase in emission signal from the acceptor fluorophorewhen in sufficient proximity to the donor fluorophore (or detecting adecrease in emission signal from the acceptor fluorophore when asignificant distance from the donor fluorophore), an increase in theemission signal from the donor fluorophore can be detected when thequencher is a significant distance from the donor fluorophore (or adecrease in emission signal from the donor fluorophore when insufficient proximity to the quencher acceptor fluorophore).

“Donor Fluorophores” are fluorophores or luminescent molecules capableof transferring energy to an acceptor fluorophore, thereby generating adetectable fluorescent signal from the acceptor. Donor fluorophores aregenerally compounds that absorb in the range of about 300 to 900 nm, forexample about 350 to 800 nm. Donor fluorophores have a strong molarabsorbance coefficient at the desired excitation wavelength, for examplegreater than about 10³ M⁻¹ cm⁻¹.

Hybridization: To form base pairs between complementary regions of twostrands of DNA, RNA, or between DNA and RNA, thereby forming a duplexmolecule. Hybridization conditions resulting in particular degrees ofstringency will vary depending upon the nature of the hybridizationmethod and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (such as the Na⁺ concentration) of the hybridization bufferwill determine the stringency of hybridization. Calculations regardinghybridization conditions for attaining particular degrees of stringencyare discussed in Sambrook et al., (1989) Molecular Cloning, secondedition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and11). The following is an exemplary set of hybridization conditions andis not limiting:

Very High Stringency (detects sequences that share at least 90%identity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (detects sequences that share at least 80% identity)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (detects sequences that share at least 60% identity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

Klebsiella pneumoniae: A gram-negative facultative anaerobic bacteriumcommonly found in the normal flora of the mouth, skin, and intestine. K.pneumoniae can cause respiratory disease, typically as a result of acolonized subject aspirating oropharyngeal bacteria into the lowerrespiratory tract. It is one of the most common causes of neonatalsepsis, especially in developing countries. Many strains of K.pneumoniae are antibiotic resistant and it is an increasingly commonnosocomial infection. Nucleic acid and protein sequences for K.pneumoniae are publicly available. For example, GenBank Accession Nos.CP000964, NC_(—)011283, NC_(—)009648, and NC_(—)012731 provide exemplaryK. pneumoniae genome sequences, all of which are incorporated byreference as provided by GenBank on Apr. 30, 2012.

The K. pneumoniae nifA gene encodes a transcriptional activator involvedin the regulation of expression of the nif genes. An exemplary K.pneumoniae nifA nucleic acid sequence includes GenBank Accession No.CP000964 (1752865-1754439), which is incorporated herein by reference aspresent in GenBank on Apr. 30, 2012. An exemplary Klebsiella pneumoniaenucleotide sequence of nifA is found at GenBank Accession No. CP000964(1752865-1754439) (SEQ ID NO: 4).

The K. pneumoniae diguanylate cyclase gene encodes an enzyme whichcatalyzes the formation of cyclic di-GMP from GTP. An exemplary K.pneumoniae diguanylate cyclase nucleic acid sequence includes GenBankAccession No. CP000964 (1413543-1415714), which is incorporated hereinby reference as present in GenBank on Feb. 15, 2013. An exemplaryKlebsiella pneumoniae nucleotide sequence of diguanylate cyclase isfound at GenBank Accession No. CP000964 (1413543-1415714) (SEQ ID NO:57).

Moraxella catarrhalis: A gram-negative bacterium (previously known asBranhamella catarrhalis) which can cause otitis media, respiratoryinfections, endocarditis, and meningitis, particularly in newborns andyoung children. Nucleic acid and protein sequences for K. pneumoniae arepublicly available. For example, GenBank Accession No. NC_(—)014147provides an exemplary M. catarrhalis genome sequence, which isincorporated by reference as provided by GenBank on Apr. 30, 2012.

The M. catarrhalis purH gene encodes a bifunctionalphosphoribosylaminoimidazolecarboxamide formyltransferase/IMPcyclohydrolase. An exemplary M. catarrhalis purH nucleic acid sequenceincludes GenBank Accession No. NC_(—)014147 (620865-622463), which isincorporated herein by reference as present in GenBank on Apr. 30, 2012.An exemplary Moraxella catarrhalis nucleotide sequence of purH is foundat GenBank Accession No. NC_(—)014147 (620865-622463) (SEQ ID NO: 5).

Neisseria meningitidis: A gram-negative bacterium that is a leadingcause of septicemia and life-threatening meningitis in children. Thereare at least five serogroups based on capsular polysaccharides (A, B, C,Y, and W135) Nucleic acid and protein sequences for N. meningitidis arepublicly available. For example, GenBank Accession Nos.CP_(—)002419-CP_(—)002424 provide exemplary N. meningitidis genomesequences, all of which are incorporated by reference as provided byGenBank on Feb. 15, 2013.

The Neisseria meningitidis sodC gene encodes a Cu,Zn superoxidedismutase. Exemplary Neisseria meningitidis sodC nucleic acid sequencesinclude GenBank Accession Nos. CP_(—)002423 (862943-493503) andCP_(—)002422 (1411887-1412447), both of which are incorporated byreference herein as provided by GenBank on Feb. 15, 2013. An exemplaryN. meningitidis sodC nucleotide sequence is found at GenBank AccessionNo. CP_(—)002423 (862943-493503) (SEQ ID NO: 61).

Pneumocystis jirovecii: Formerly classified as Pneumocystis carinii. Anonfilamentous fungus that can cause severe pneumonia inimmunocompromised patients and neonates. P. jirovecii may also colonizethe lungs of healthy individuals without causing disease. Nucleic acidand protein sequences for P. jirovecii are publicly available.

The P. jirovecii dhps gene encodes a diydropteroate synthase. Anexemplary P. jirovecii dhps nucleic acid sequence includes GenBankAccession No. AF139132, which is incorporated herein by reference aspresent in GenBank on Apr. 30, 2012. An exemplary Pneumocystis jiroveciinucleotide sequence of dhps is found at GenBank Accession No. AF139132(SEQ ID NO: 6).

Pseudomonas aeruginosa: A gram-negative bacterium that is a leadingcause of hospital-acquired infections, including bacteremia, burn/woundinfections, and severe pneumonia, including in neonatal intensive careunits. This organism is also a common cause of community-acquired skin,ear, and eye infections, often associated with swimming in contaminatedrecreational facilities. Nucleic acid and protein sequences for P.aeruginosa are publicly available. For example, GenBank Accession Nos.NC_(—)011770, NC_(—)009656, NC_(—)002516, and NC_(—)008463 provideexemplary P. aeruginosa genome sequences, all of which are incorporatedby reference as provided by GenBank on Apr. 30, 2012.

The P. aeruginosa gyrb gene encodes a dna gyrase subunit B. Exemplary P.aeruginosa gyrb nucleic acid sequences include GenBank Accession Nos.AB005881, NC_(—)002516 (4275-6695), NC_(—)009656 (4274-6694), andNC_(—)008463 (4275-6695), which are incorporated herein by reference aspresent in GenBank on Apr. 30, 2012. An exemplary Pseudomonas aeruginosanucleotide sequence of gyrb is found at GenBank Accession No. AB005881(SEQ ID NO: 7).

Shigella: A genus of Gram-negative, nonspore forming, non-motile,rod-shaped bacteria closely related to Escherichia coli and Salmonella.The causative agent of human shigellosis, Shigella causes disease inprimates, but not in other mammals. It can cause infection in neonatesas a result of maternal transmission during delivery. During infection,it typically causes dysentery. Phylogenetic studies indicate thatShigella may be more appropriately treated as subgenus of Escherichia.

Staphylococcus aureus: A gram-positive bacterium which can causediseases including skin infection, respiratory infection, meningitis,endocarditis, toxic shock syndrome, and sepsis. It is one of the mostcommon nosocomial infections and is increasing in frequency in neonatalintensive care units. Multi-drug resistant strains (includingmethicillin-resistant S. aureus; MRSA) are increasingly common. Nucleicacid and protein sequences for S. aureus are publicly available. Forexample, GenBank Accession Nos. NC_(—)007622, NC_(—)002951,NC_(—)013450, and NC_(—)009632 provide exemplary S. aureus genomesequences, all of which are incorporated by reference as provided byGenBank on Apr. 30, 2012.

The S. aureus gsf gene encodes a conserved region in glutamate synthasefamily protein. An exemplary S. aureus gsf nucleic acid sequenceincludes GenBank Accession No. CP003194 (2567647-2569224), which isincorporated by reference as provided by GenBank on Apr. 30, 2012. Anexemplary Staphylococcus aureus nucleotide sequence of gsf is found atGenBank Accession No. CP003194 (2567647-2569224) (SEQ ID NO: 8).

Streptococcus agalactiae (Group B Streptococcus; GBS): A gram-positivebacterium that is a major cause of meningitis and sepsis in neonates.GBS can also asymptomatically colonize skin and mucous membranes.Nucleic acid and protein sequences for GBS are publicly available. Forexample, GenBank Accession Nos. NC_(—)007432, NC_(—)004166, andNC_(—)019048 provide exemplary GBS genome sequences, all of which areincorporated by reference as provided by GenBank on Feb. 15, 2013.

The GBS cfb gene encodes the CAMP factor. Exemplary GBS cfb nucleic acidsequences include GenBank Accession Nos. NC_(—)004116 (2016473-2017240),NC_(—)007432 (1969227-1969994), and NC_(—)019048 (1695124-1695891), allof which are incorporated by reference herein as provided by GenBank onFeb. 15, 2013. An exemplary GBS nucleotide sequence of cfb is found atGenBank Accession No. NC_(—)004116 (2016473-2017240) (SEQ ID NO: 53).

Toxoplasma gondii: A parasite of many animal species. Cats are the onlyhost capable of passing infective T. gondii oocysts to subsequent hosts.Humans may become infected by exposure to contaminated undercooked meator exposure to water, soil, or other material contaminated with T.gondii laden feline feces. Infection may be asymptomatic or elicit mildsymptoms; however serious neurologic or ocular disease may result in thefetus of an exposed pregnant women. Nucleic acid and protein sequencesfor T. gondii are publicly available. For example, GenBank Accession No.NZ_ABPA00000000 provides an exemplary T. gondii genome sequence, whichis incorporated by reference as provided by GenBank on Apr. 30, 2012.

The T. gondii ssrRNA gene is a small subunit ribosomal RNA. An exemplaryT. gondii ssrRNA nucleic acid sequence includes GenBank Accession No.EF472967, which is incorporated herein by reference as present inGenBank on Apr. 30, 2012. An exemplary Toxoplasma gondii nucleotidesequence of ssrRNA is found at GenBank Accession No. EF472967 (SEQ IDNO: 9).

Ureaplasma parvum: Previously classified as Ureaplasma urealyticumbiovar 1. A mycoplasma which can cause genito-urinary infection andinfertility. It can also cause respiratory infection in neonates as aresult of maternal transmission during delivery. Nucleic acid andprotein sequences for U. parvum are publicly available. For example,GenBank Accession Nos. NC_(—)010503 and NC_(—)002162 provide exemplaryU. parvum genome sequences, both of which are incorporated by referenceas provided by GenBank on Apr. 30, 2012.

The U. parvum ure gene encodes the urease complex. An exemplary U.parvum ure nucleic acid sequence includes GenBank Accession No.AF085733, which is incorporated by reference as provided by GenBank onApr. 30, 2012. An exemplary Ureaplasma parvum nucleotide sequence ofureC is found at GenBank Accession No. AF085733 (912-2708) (SEQ ID NO:10).

Ureaplasma spp.: A genus of gram-negative bacteria which is ureasepositive. Ureaplasma spp. refers to any species in the genus Ureaplasma.In some embodiments, Ureaplasma spp. includes Ureaplasma parvum andUreaplasma urealyticum. One of ordinary skill in the art can identifyadditional Ureaplasma spp. (such as U. canigenitalium, U. cati, U.diversum, U. felinum, U. gallorale, and U. loridis), for exampleutilizing the NCBI Taxonomy Browser (e.g.,www.ncbi.nlm.nih.gov/Taxonomy/).

Ureaplasma urealyticum: A mycoplasma which can cause genito-urinaryinfection, infertility, and meningitis. It can also cause respiratorydisease in neonates as a result of maternal transmission duringdelivery. Nucleic acid and protein sequences for U. urealyticum arepublicly available. For example, GenBank Accession No. NC_(—)011374provides an exemplary U. urealyticum genome sequence, which isincorporated by reference as provided by GenBank on Apr. 30, 2012.

The U. urealyticum mba gene encodes the multiple banded antigen. Anexemplary U. urealyticum mba nucleic acid sequence includes GenBankAccession No. AF055367, which is incorporated by reference as providedby GenBank on Apr. 30, 2012. An exemplary Ureaplasma urealyticumnucleotide sequence of mba is found at GenBank Accession No. AF055367(SEQ ID NO: 11).

III. Methods for Detection of Pathogens

Methods for detecting the presence of a pathogen such as Acinetobacterbaumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasmagondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp. Streptococcus agalactiae, and/or Neisseria meningitidis in a sampleare disclosed, for example, utilizing the probes and/or primersdisclosed herein. In some embodiments, the methods include detection ofa single selected pathogen. In other embodiments, the methods includedetection of one or more of Acinetobacter baumannii, Pseudomonasaeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxellacatarrhalis, Escherichia coli, Shigella, Staphylococcus aureus,Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum,Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcusagalactiae, Neisseria meningitidis or any combination of two or morethereof (for example, utilizing a simultaneous multipathogen detectionassay (such as an array or card assay) or a multiplex assay).

The methods described herein may be used for any purpose for whichdetection of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellapneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli,Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydiatrachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp.,Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidisis desirable, including diagnostic or prognostic applications, such asin laboratory or clinical settings.

Appropriate samples include any conventional environmental or biologicalsamples, including clinical samples obtained from a human or veterinarysubject. Suitable samples include all biological samples useful fordetection of infection in subjects, including, but not limited to,cells, tissues (for example, lung, liver, or kidney), autopsy samples,bone marrow aspirates, bodily fluids (for example, blood, serum, urine,cerebrospinal fluid, middle ear fluids, bronchoalveolar lavage, trachealaspirates, sputum, nasopharyngeal swabs or aspirates, oropharyngealswabs or aspirates, or saliva), eye swabs, cervical swabs, vaginalswabs, rectal swabs, stool, and stool suspensions. Suitable samples alsoinclude all samples useful for detection of a pathogen in an environment(such as a clinic or hospital), including but not limited to a water orfluid sample, a food sample, or a surface swab (for example, a swab of acounter, bed, floor, wall, or other surface). Standard techniques foracquisition of such samples are available. See for example, Schluger etal., J. Exp. Med. 176:1327-1333, 1992; Bigby et al., Am. Rev. Respir.Dis. 133:515-518, 1986; Kovacs et al., N. Engl. J. Med. 318:589-593,1988; and Ognibene et al., Am. Rev. Respir. Dis. 129:929-932, 1984.

In some embodiments, the nucleic acids detected using the methodsprovided herein include nucleic acid molecules from Acinetobacterbaumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasmagondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, or Neisseria meningitidis. In at leastsome embodiments, the disclosed methods can detect multiple strains orserotypes of a pathogen species. In some examples, the nucleic acidsdetected include nucleic acids from multidrug-resistant strains ofAcinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, and/or Neisseria meningitidis. Strainsof particular pathogens may be obtained from patient or environmentalsamples or laboratory or reference collections, for example, theAmerican Type Culture Collection (Manassas, Va.). In one non-limitingexample, the disclosed methods detect nucleic acids frommethicillin-resistant S. aureus (MRSA), including hospital-acquired MRSA(HA-MRSA) or community-acquired MRSA (CA-MRSA). In other non-limitingexamples, the disclosed methods detect nucleic acids from E. colivirotypes including enterotoxigenic E. coli (ETEC), enteroinvasive E.coli (EIEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli(EPEC), or enteroaggregative E. coli (EAEC).

One of ordinary skill in the art will know suitable methods forextracting nucleic acids such as RNA and/or DNA from a sample; suchmethods will depend upon, for example, the type of sample in which thepathogen nucleic acid is found. Nucleic acids can be extracted usingstandard methods. For instance, rapid nucleic acid preparation can beperformed using a commercially available kit (such as kits and/orinstruments from Qiagen (such as DNEASY® or RNEASY® kits), Roche AppliedScience (such as MAGNA PURE® kits and instruments), Thermo Scientific(KingFisher mL), bioMérieux (NUCLISENS® NASBA Diagnostics), or Epicentre(MASTERPURE™ kits)). In other examples, the nucleic acids may beextracted using guanidinium isothiocyanate, such as single-stepisolation by acid guanidinium isothiocyanate-phenol-chloroformextraction (Chomczynski et al. Anal. Biochem. 162:156-159, 1987). Thesample can be used directly or can be processed, such as by addingsolvents, preservatives, buffers, or other compounds or substances.

Detecting presence of at least one of an Acinetobacter baumannii,Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii,Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcusaureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasmaurealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp.,Streptococcus agalactiae, or Neisseria meningitidis nucleic acid in asample involves contacting the sample with at least one of the probes(such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 probes)disclosed herein that is capable of hybridizing to an Acinetobacterbaumannii oxa-51 nucleic acid, a Pseudomonas aeruginosa gyrB nucleicacid, a Klebsiella pneumoniae nifA nucleic acid, a Toxoplasma gondiissrRNA nucleic acid, a Moraxella catarrhalis purH nucleic acid, anEscherichia coli and/or Shigella uidA nucleic acid, a Staphylococcusaureus gsf nucleic acid, a Pneumocystis jirovecii dhps nucleic acid, aChlamydia trachomatis tmRNA nucleic acid, a Ureaplasma urealyticum mbanucleic acid, a Ureaplasma parvum ure nucleic acid, a Ureaplasma spp.ure nucleic acid, a Bartonella spp. ssrA nucleic acid, a Streptococcusagalactiae cfb nucleic acid, or a Neisseria meningitidis sodC nucleicacid, for example, under conditions of high or very high stringency.

One of ordinary skill in the art can determine low, high, or very highstringency conditions for hybridization of a primer or probe (such as aprobe or primer disclosed herein) to a nucleic acid sequence (forexample to one of SEQ ID NOs: 1-11, 52, 53, 57, or 61). In someexamples, the conditions are for hybridization of a primer or probe to anucleic acid attached to a solid support (such as the conditionsprovided above). In other examples, the conditions are for hybridizationof a primer or probe to a nucleic acid in solution, such as a PCRreaction mixture. In some non-limiting examples, low stringencyconditions include hybridization (such as an annealing step in PCR) at atemperature of about 45-50° C. In other examples, high stringencyconditions include hybridization (such as an annealing step in PCR) at atemperature of about 50-60° C. In further examples, very high stringencyconditions include hybridization (such as an annealing step in PCR) at atemperature of greater than 60° C. One of skill in the art can determineappropriate hybridization or annealing conditions (including the degreeof hybridization) based on the particular primers or probes and targetnucleic acids to be amplified or detected.

In some embodiments, the methods include contacting the sample with atleast one probe comprising a nucleic acid molecule between 10 and 40nucleotides in length and detecting hybridization between the one ormore probes and a nucleic acid in the sample, wherein detection ofhybridization indicates the presence of one or more of said pathogens inthe sample. In some examples, the probe is capable of hybridizing (suchas under high stringency or very high stringency conditions) to anAcinetobacter baumannii nucleic acid sequence set forth as SEQ ID NO: 1,a Chlamydia trachomatis nucleic acid sequence set forth as SEQ ID NO: 2,an Escherichia coli nucleic acid sequence set forth as SEQ ID NO: 3, aKlebsiella pneumoniae nucleic acid sequence set forth as SEQ ID NO: 4 orSEQ ID NO: 57, a Moraxella catarrhalis nucleic acid sequence set forthas SEQ ID NO: 5, a Pneumocystis jirovecii nucleic acid sequence setforth as SEQ ID NO: 6, a Pseudomonas aeruginosa nucleic acid sequenceset forth as SEQ ID NO: 7, a Staphylococcus aureus nucleic acid sequenceset forth as SEQ ID NO: 8, a Toxoplasma gondii nucleic acid sequence setforth as SEQ ID NO: 9, a Ureaplasma parvum nucleic acid sequence setforth as SEQ ID NO: 10, a Ureaplasma urealyticum nucleic acid sequenceset forth as SEQ ID NO: 11, a Bartonella spp. nucleic acid set forth asSEQ ID NO: 52, a Streptococcus agalactiae nucleic acid sequence setforth as SEQ ID NO: 53, a Neisseria meningitidis nucleic acid set forthas SEQ ID NO: 61, or a nucleic acid sequence at least 90% identical (forexample 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%identical) to one of SEQ ID NOs: 1-11 52-53, 57, or 61. In someexamples, the sample is contacted with one or more nucleic acid probesbetween 20 and 40 nucleotides in length comprising or consisting of anucleic acid sequence set forth as any one of SEQ ID NOs: 14, 17, 20,23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 56, 60, 64, or the reversecomplement thereof.

In particular examples, the probes are detectably labeled (for example,as described in section IV, below). In some examples, the probes are atleast 10, 15, 20, 25, 30, 35, or 40 nucleotides in length. In otherexamples, the probes may be no more than 10, 15, 20, 25, 30, 35, or 40nucleotides in length. In further examples, the probes are 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

Detection of hybridization between an Acinetobacter baumannii probe (forexample SEQ ID NO: 14) and a nucleic acid indicates the presence ofAcinetobacter baumannii nucleic acid in the sample, detection ofhybridization between a Chlamydia trachomatis probe (for example SEQ IDNO: 38) and a nucleic acid indicates the presence of Chlamydiatrachomatis nucleic acid in the sample, detection of hybridizationbetween an Escherichia coli/Shigella probe (for example SEQ ID NO: 29)and a nucleic acid indicates the presence of E. coli and/or Shigellanucleic acid in the sample, detection of hybridization between aKlebsiella pneumoniae probe (for example SEQ ID NO: 20 or SEQ ID NO: 60)and a nucleic acid indicates the presence of Klebsiella pneumoniaenucleic acid in the sample, detection of hybridization between aMoraxella catarrhalis probe (for example SEQ ID NO: 26) and a nucleicacid indicates the presence of Moraxella catarrhalis nucleic acid in thesample, detection of hybridization between a Pneumocystis jiroveciiprobe (for example SEQ ID NO: 35) and a nucleic acid indicates thepresence of Pneumocystis jirovecii nucleic acid in the sample, detectionof hybridization between a Pseudomonas aeruginosa probe (for example SEQID NO: 17) and a nucleic acid indicates the presence of Pseudomonasaeruginosa nucleic acid in the sample, detection of hybridizationbetween a Staphylococcus aureus probe (for example SEQ ID NO: 32) and anucleic acid indicates the presence of Staphylococcus aureus nucleicacid in the sample, detection of hybridization between a Toxoplasmagondii probe (for example SEQ ID NO: 23) and a nucleic acid indicatesthe presence of Toxoplasma gondii nucleic acid in the sample, detectionof hybridization between a Ureaplasma parvum probe (for example SEQ IDNO: 44) and a nucleic acid indicates the presence of Ureaplasma parvumnucleic acid in the sample, detection of hybridization between aUreaplasma urealyticum probe (for example SEQ ID NO: 41) and a nucleicacid indicates the presence of Ureaplasma urealyticum nucleic acid inthe sample, detection of hybridization between a Ureaplasma spp. probe(for example SEQ ID NO: 47) and a nucleic acid indicates the presence ofUreaplasma spp. nucleic acid in the sample, detection of hybridizationbetween a Bartonella spp. probe (for example SEQ ID NO: 50) and anucleic acid indicates the presence of Bartonella spp. nucleic acid inthe sample, detection of hybridization between a Streptococcusagalactiae probe (for example SEQ ID NO: 56) and a nucleic acidindicates the presence of Streptococcus agalactiae nucleic acid in thesample, and detection of hybridization between a Neisseria meningitidisprobe (for example SEQ ID NO: 64) and a nucleic acid indicates thepresence of a Neisseria meningitidis nucleic acid in the sample.

In some embodiments, the methods disclosed herein further includepositive and/or negative controls. One of ordinary skill in the art canselect suitable controls. In some examples, a negative control is a notemplate control (such as a reaction that includes all components exceptthe nucleic acid sample). In other examples, a positive control includesa sample known to include nucleic acid from a particular pathogen. Infurther examples, a positive control includes an internal positivecontrol, such as a human nucleic acid (for example, RNase P) when thesample is from a human subject. In other examples, a positive controlincludes a synthetic positive control (such as a combined positivecontrol), for example, a nucleic acid molecule including forward primer,probe, and reverse primer sequences for one or more primer/probe setsincluded in the assay. A combined positive control may also includeadditional positive or negative controls, such as a human nucleic acidcontrol (for example, RNase P), and/or a control for laboratorycontamination (such as a primer/probe that is not included in theassay). See, e.g., Kodani and Winchell (J. Clin. Microbiol.50:1057-1060, 2011; incorporated herein by reference) for exemplarymethods for constructing a combined positive control.

In some embodiments, nucleic acids present in a sample (for example, oneor more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellapneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli,Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydiatrachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp.,Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidisnucleic acids) are amplified prior to using a probe for detection. Forinstance, it can be advantageous to amplify a portion of one of more ofthe disclosed nucleic acids, and then detect the presence of theamplified nucleic acid, for example, to increase the number of nucleicacids that can be detected, thereby increasing the signal obtained.Specific nucleic acid primers can be used to amplify a region that is atleast about 50, at least about 60, at least about 70, at least about 80at least about 90, at least about 100, at least about 200, at leastabout 250, at least about 300, at least about 400, at least about 500,at least about 1000, at least about 2000, or more base pairs in lengthto produce amplified nucleic acids (such as amplified Acinetobacterbaumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasmagondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, or Neisseria meningitidis nucleicacids). In other examples, specific nucleic acid primers can be used toamplify a region that is about 50-3000 base pairs in length (forexample, about 70-2000 base pairs, about 100-1000 base pairs, about50-300 base pairs, about 300-500 base pairs, or about 1000-3000 basepairs in length).

Detecting the amplified product typically includes the use of labeledprobes that are sufficiently complementary to, and hybridize to, theamplified nucleic acid sequence. Thus, the presence, amount, and/oridentity of the amplified product can be detected by hybridizing alabeled probe, such as a fluorescently labeled probe, complementary tothe amplified product. In one embodiment, the detection of a targetnucleic acid sequence of interest, such as an Acinetobacter baumanniioxa-51 nucleic acid, a Pseudomonas aeruginosa gyrB nucleic acid, aKlebsiella pneumoniae nifA nucleic acid, a Klebsiella pneumoniaediguanylate cyclase nucleic acid, a Toxoplasma gondii ssrRNA nucleicacid, a Moraxella catarrhalis purH nucleic acid, an Escherichia coliand/or Shigella uidA nucleic acid, a Staphylococcus aureus gsf nucleicacid, a Pneumocystis jirovecii dhps nucleic acid, a Chlamydiatrachomatis tmRNA nucleic acid, a Ureaplasma urealyticum mba nucleicacid, a Ureaplasma parvum ure nucleic acid, a Ureaplasma spp. urenucleic acid, a Bartonella spp. ssrA nucleic acid, a Streptococcusagalactiae cfb nucleic acid, or a Neisseria meningitidis sodC nucleicacid includes the combined use of PCR amplification and a labeled probesuch that the product is measured using real-time PCR (such as TAQMAN®real-time PCR). In another embodiment, the detection of an amplifiedtarget nucleic acid sequence of interest includes the transfer of theamplified target nucleic acid to a solid support, such as a blot, forexample a Northern blot, and probing the blot with a probe, for examplea labeled probe, that is complementary to the amplified target nucleicacid. In still further embodiments, the detection of amplified targetnucleic acid of interest includes the hybridization of a labeledamplified target nucleic acid to probes disclosed herein that arearrayed in a predetermined array with an addressable location and thatare complementary to the amplified target nucleic acid.

Any nucleic acid amplification method can be used to detect the presenceof one or more of Acinetobacter baumannii, Pseudomonas aeruginosa,Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis,Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystisjirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasmaparvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, orNeisseria meningitidis nucleic acids in a sample. In one specific,non-limiting example, polymerase chain reaction (PCR) is used to amplifythe pathogen-specific nucleic acid sequences. In other specific,non-limiting examples, real-time PCR, reverse transcriptase-polymerasechain reaction (RT-PCR), real-time reverse transcriptase-polymerasechain reaction (rt RT-PCR), ligase chain reaction, ortranscription-mediated amplification (TMA) is used to amplify thenucleic acids. In a specific example, one or more (such as 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of an Acinetobacter baumanniioxa-51 nucleic acid, a Pseudomonas aeruginosa gyrB nucleic acid, aKlebsiella pneumoniae nifA nucleic acid, a Klebsiella pneumoniaediguanylate cyclase nucleic acid, a Toxoplasma gondii ssrRNA nucleicacid, a Moraxella catarrhalis purH nucleic acid, an Escherichiacoli/Shigella uidA nucleic acid, a Staphylococcus aureus gsf nucleicacid, a Pneumocystis jirovecii dhps nucleic acid, a Chlamydiatrachomatis tmRNA nucleic acid, a Ureaplasma urealyticum mba nucleicacid, a Ureaplasma parvum ure nucleic acid, a Ureaplasma spp. urenucleic acid, a Bartonella spp. ssrA nucleic acid, a Streptococcusagalactiae cfb nucleic acid, a Neisseria meningitidis sodC nucleic acid,or any combination of two or more thereof are amplified by real-timePCR, for example real-time TAQMAN® PCR. Techniques for nucleic acidamplification are well-known to those of ordinary skill in the art.

Typically, at least two primers are utilized in the amplificationreaction. In some examples, amplification of an Acinetobacter baumanniinucleic acid involves contacting the Acinetobacter baumannii nucleicacid with one or more primers (such as two or more primers) that arecapable of hybridizing to and directing the amplification of anAcinetobacter baumannii nucleic acid, such as a primer capable ofhybridizing under high or very high stringency conditions to anAcinetobacter baumannii nucleic acid sequence set forth as SEQ NO: 1,for example a primer that is least 90% identical (such as 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotidesequence set forth as one of SEQ ID NOs: 12 or 13. In one example, anAcinetobacter baumannii oxa-51 nucleic acid is amplified utilizing apair of primers, such as a forward primer at least 90% identical to SEQID NO: 12 and a reverse primer at least 90% identical to SEQ ID NO: 13,such as a forward primer comprising or consisting essentially of SEQ IDNO: 12 and a reverse primer comprising or consisting essentially of SEQID NO: 13.

In other examples, amplification of a Chlamydia trachomatis nucleic acidinvolves contacting the Chlamydia trachomatis nucleic acid with one ormore primers (such as two or more primers) that are capable ofhybridizing to and directing the amplification of a Chlamydiatrachomatis nucleic acid, such as a primer capable of hybridizing underhigh or very high stringency conditions to a Chlamydia trachomatisnucleic acid sequence set forth as SEQ NO: 2, for example a primer thatis least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical) to the nucleotide sequence set forth as one ofSEQ ID NOs: 36 or 37. In one example, a Chlamydia trachomatis tmRNAnucleic acid is amplified utilizing a pair of primers, such as a forwardprimer at least 90% identical to SEQ ID NO: 36 and a reverse primer atleast 90% identical to SEQ ID NO: 37, such as a forward primercomprising or consisting essentially of SEQ ID NO: 36 and a reverseprimer comprising or consisting essentially of SEQ ID NO: 37.

In further examples, amplification of an Escherichia coli and/orShigella nucleic acid involves contacting the Escherichia coli and/orShigella nucleic acid with one or more primers (such as two or moreprimers) that are capable of hybridizing to and directing theamplification of an Escherichia coli and/or Shigella nucleic acid, suchas a primer capable of hybridizing under high or very high stringencyconditions to an Escherichia coli and/or Shigella nucleic acid sequenceset forth as SEQ NO: 3, for example a primer that is least 90% identical(such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical)to the nucleotide sequence set forth as one of SEQ ID NOs: 27 or 28. Inone example, an Escherichia coli uidA nucleic acid is amplifiedutilizing a pair of primers, such as a forward primer at least 90%identical to SEQ ID NO: 27 and a reverse primer at least 90% identicalto SEQ ID NO: 28, such as a forward primer comprising or consistingessentially of SEQ ID NO: 27 and a reverse primer comprising orconsisting essentially of SEQ ID NO: 28.

In some examples, amplification of a Klebsiella pneumoniae nucleic acidinvolves contacting the Klebsiella pneumoniae nucleic acid with one ormore primers (such as two or more primers) that are capable ofhybridizing to and directing the amplification of a Klebsiellapneumoniae nucleic acid, such as a primer capable of hybridizing underhigh or very high stringency conditions to a Klebsiella pneumoniaenucleic acid sequence set forth as SEQ NO: 4, for example a primer thatis least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical) to the nucleotide sequence set forth as one ofSEQ ID NOs: 18 or 19. In one example, a Klebsiella pneumoniae nifAnucleic acid is amplified utilizing a pair of primers, such as a forwardprimer at least 90% identical to SEQ ID NO: 18 and a reverse primer atleast 90% identical to SEQ ID NO: 19, such as a forward primercomprising or consisting essentially of SEQ ID NO: 18 and a reverseprimer comprising or consisting essentially of SEQ ID NO: 19. In otherexamples, amplification of a Klebsiella pneumoniae nucleic acid involvescontacting the Klebsiella pneumoniae nucleic acid with one or moreprimers (such as two or more primers) that are capable of hybridizing toand directing the amplification of a Klebsiella pneumoniae nucleic acid,such as a primer capable of hybridizing under high or very highstringency conditions to a Klebsiella pneumoniae nucleic acid sequenceset forth as SEQ NO: 57, for example a primer that is least 90%identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 58or 59. In one example, a Klebsiella pneumoniae diguanylate cyclasenucleic acid is amplified utilizing a pair of primers, such as a forwardprimer at least 90% identical to SEQ ID NO: 58 and a reverse primer atleast 90% identical to SEQ ID NO: 59, such as a forward primercomprising or consisting essentially of SEQ ID NO: 58 and a reverseprimer comprising or consisting essentially of SEQ ID NO: 59.

In additional examples, amplification of a Moraxella catarrhalis nucleicacid involves contacting the Moraxella catarrhalis nucleic acid with oneor more primers (such as two or more primers) that are capable ofhybridizing to and directing the amplification of a Moraxellacatarrhalis nucleic acid, such as a primer capable of hybridizing underhigh or very high stringency conditions to a Moraxella catarrhalisnucleic acid sequence set forth as SEQ NO: 5, for example a primer thatis least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical) to the nucleotide sequence set forth as one ofSEQ ID NOs: 24 or 25. In one example, a Moraxella catarrhalis purHnucleic acid is amplified utilizing a pair of primers, such as a forwardprimer at least 90% identical to SEQ ID NO: 24 and a reverse primer atleast 90% identical to SEQ ID NO: 25, such as a forward primercomprising or consisting essentially of SEQ ID NO: 24 and a reverseprimer comprising or consisting essentially of SEQ ID NO: 25.

In additional examples, amplification of a Pneumocystis jiroveciinucleic acid involves contacting the Pneumocystis jirovecii nucleic acidwith one or more primers (such as two or more primers) that are capableof hybridizing to and directing the amplification of a Pneumocystisjirovecii nucleic acid, such as a primer capable of hybridizing underhigh or very high stringency conditions to a Pneumocystis jiroveciinucleic acid sequence set forth as SEQ NO: 6, for example a primer thatis least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical) to the nucleotide sequence set forth as one ofSEQ ID NOs: 33 or 34. In one example, a P. jirovecii dhps nucleic acidis amplified utilizing a pair of primers, such as a forward primer atleast 90% identical to SEQ ID NO: 33 and a reverse primer at least 90%identical to SEQ ID NO: 34, such as a forward primer comprising orconsisting essentially of SEQ ID NO: 33 and a reverse primer comprisingor consisting essentially of SEQ ID NO: 34.

In further examples, amplification of a Pseudomonas aeruginosa nucleicacid involves contacting the Pseudomonas aeruginosa nucleic acid withone or more primers (such as two or more primers) that are capable ofhybridizing to and directing the amplification of a Pseudomonasaeruginosa nucleic acid, such as a primer capable of hybridizing underhigh or very high stringency conditions to a Pseudomonas aeruginosanucleic acid sequence set forth as SEQ NO: 7, for example a primer thatis least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical) to the nucleotide sequence set forth as one ofSEQ ID NOs: 15 or 16. In one example, a Pseudomonas aeruginosa gyrBnucleic acid is amplified utilizing a pair of primers, such as a forwardprimer at least 90% identical to SEQ ID NO: 15 and a reverse primer atleast 90% identical to SEQ ID NO: 16, such as a forward primercomprising or consisting essentially of SEQ ID NO: 15 and a reverseprimer comprising or consisting essentially of SEQ ID NO: 16.

In still further examples, amplification of a Staphylococcus aureusnucleic acid involves contacting the Staphylococcus aureus nucleic acidwith one or more primers (such as two or more primers) that are capableof hybridizing to and directing the amplification of a Staphylococcusaureus nucleic acid, such as a primer capable of hybridizing under highor very high stringency conditions to a Staphylococcus aureus nucleicacid sequence set forth as SEQ NO: 8, for example a primer that is least90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical) to the nucleotide sequence set forth as one of SEQ IDNOs: 30 or 31. In one example, a Staphylococcus aureus gsf nucleic acidis amplified utilizing a pair of primers, such as a forward primer atleast 90% identical to SEQ ID NO: 30 and a reverse primer at least 90%identical to SEQ ID NO: 31, such as a forward primer comprising orconsisting essentially of SEQ ID NO: 30 and a reverse primer comprisingor consisting essentially of SEQ ID NO: 31.

In other examples, amplification of a Toxoplasma gondii nucleic acidinvolves contacting the Toxoplasma gondii nucleic acid with one or moreprimers (such as two or more primers) that are capable of hybridizing toand directing the amplification of a Toxoplasma gondii nucleic acid,such as a primer capable of hybridizing under high or very highstringency conditions to a Toxoplasma gondii nucleic acid sequence setforth as SEQ NO: 9, for example a primer that is least 90% identical(such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical)to the nucleotide sequence set forth as one of SEQ ID NOs: 21 or 22. Inone example, a Toxoplasma gondii ssrRNA nucleic acid is amplifiedutilizing a pair of primers, such as a forward primer at least 90%identical to SEQ ID NO: 21 and a reverse primer at least 90% identicalto SEQ ID NO: 22, such as a forward primer comprising or consistingessentially of SEQ ID NO: 21 and a reverse primer comprising orconsisting essentially of SEQ ID NO: 22.

In additional examples, amplification of a Ureaplasma parvum nucleicacid involves contacting the Ureaplasma parvum nucleic acid with one ormore primers (such as two or more primers) that are capable ofhybridizing to and directing the amplification of a Ureaplasma parvumnucleic acid, such as a primer capable of hybridizing under high or veryhigh stringency conditions to a Ureaplasma parvum nucleic acid sequenceset forth as SEQ NO: 10, for example a primer that is least 90%identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 42or 43. In one example, a Ureaplasma parvum ure nucleic acid is amplifiedutilizing a pair of primers, such as a forward primer at least 90%identical to SEQ ID NO: 42 and a reverse primer at least 90% identicalto SEQ ID NO: 43, such as a forward primer comprising or consistingessentially of SEQ ID NO: 42 and a reverse primer comprising orconsisting essentially of SEQ ID NO: 43.

In further examples, amplification of a Ureaplasma urealyticum nucleicacid involves contacting the Ureaplasma urealyticum nucleic acid withone or more primers (such as two or more primers) that are capable ofhybridizing to and directing the amplification of a Ureaplasmaurealyticum nucleic acid, such as a primer capable of hybridizing underhigh or very high stringency conditions to a Ureaplasma urealyticumnucleic acid sequence set forth as SEQ NO: 11, for example a primer thatis least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical) to the nucleotide sequence set forth as one ofSEQ ID NOs: 39 or 40. In one example, a Ureaplasma urealyticum mbanucleic acid is amplified utilizing a pair of primers, such as a forwardprimer at least 90% identical to SEQ ID NO: 39 and a reverse primer atleast 90% identical to SEQ ID NO: 40, such as a forward primercomprising or consisting essentially of SEQ ID NO: 39 and a reverseprimer comprising or consisting essentially of SEQ ID NO: 40.

In additional examples, amplification of a Ureaplasma spp. nucleic acidinvolves contacting the Ureaplasma spp. nucleic acid with one or moreprimers (such as two or more primers) that are capable of hybridizing toand directing the amplification of a Ureaplasma spp. nucleic acid, suchas a primer capable of hybridizing under very high stringency conditionsto a Ureaplasma spp. nucleic acid sequence set forth as SEQ NO: 10, forexample a primer that is least 90% identical (such as 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotidesequence set forth as one of SEQ ID NOs: 45 or 46. In one example, aUreaplasma spp. ure nucleic acid is amplified utilizing a pair ofprimers, such as a forward primer at least 90% identical to SEQ ID NO:45 and a reverse primer at least 90% identical to SEQ ID NO: 46, such asa forward primer comprising or consisting essentially of SEQ ID NO: 45and a reverse primer comprising or consisting essentially of SEQ ID NO:46.

In further examples, amplification of a Bartonella spp. nucleic acidinvolves contacting the Bartonella spp. nucleic acid with one or moreprimers (such as two or more primers) that are capable of hybridizing toand directing the amplification of a Bartonella spp. nucleic acid, suchas a primer capable of hybridizing under high or very high stringencyconditions to a Bartonella spp. ssrA nucleic acid sequence (such as SEQID NO: 52), for example a primer that is least 90% identical (such as91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to thenucleotide sequence set forth as one of SEQ ID NOs: 48, 49, or 51. Inone example, a Bartonella spp. ssrA nucleic acid is amplified utilizinga pair of primers, such as a forward primer at least 90% identical toSEQ ID NO: 48 and a reverse primer at least 90% identical to SEQ ID NO:49, such as a forward primer comprising or consisting essentially of SEQID NO: 48 and a reverse primer comprising or consisting essentially ofSEQ ID NO: 49. In other examples, a Bartonella spp. ssrA nucleic acid isamplified utilizing a pair of primers, such as a forward primer at least90% identical to SEQ ID NO: 51 and a reverse primer at least 90%identical to SEQ ID NO: 49, such as a forward primer comprising orconsisting essentially of SEQ ID NO: 51 and a reverse primer comprisingor consisting essentially of SEQ ID NO: 49.

In other examples, amplification of a Streptococcus agalactiae nucleicacid involves contacting the Streptococcus agalactiae nucleic acid withone or more primers (such as two or more primers) that are capable ofhybridizing to and directing the amplification of a Streptococcusagalactiae nucleic acid, such as a primer capable of hybridizing underhigh or very high stringency conditions to a Streptococcus agalactiaenucleic acid sequence set forth as SEQ NO: 53, for example a primer thatis least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical) to the nucleotide sequence set forth as one ofSEQ ID NOs: 54 or 55. In one example, a Streptococcus agalactiae cfbnucleic acid is amplified utilizing a pair of primers, such as a forwardprimer at least 90% identical to SEQ ID NO: 54 and a reverse primer atleast 90% identical to SEQ ID NO: 55, such as a forward primercomprising or consisting essentially of SEQ ID NO: 54 and a reverseprimer comprising or consisting essentially of SEQ ID NO: 55.

In other examples, amplification of a Neisseria meningitidis nucleicacid involves contacting the Neisseria meningitidis nucleic acid withone or more primers (such as two or more primers) that are capable ofhybridizing to and directing the amplification of a Neisseriameningitidis nucleic acid, such as a primer capable of hybridizing underhigh or very high stringency conditions to a Neisseria meningitidisnucleic acid sequence set forth as SEQ NO: 61, for example a primer thatis least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical) to the nucleotide sequence set forth as one ofSEQ ID NOs: 62 or 63. In one example, a Neisseria meningitidis sodCnucleic acid is amplified utilizing a pair of primers, such as a forwardprimer at least 90% identical to SEQ ID NO: 62 and a reverse primer atleast 90% identical to SEQ ID NO: 63, such as a forward primercomprising or consisting essentially of SEQ ID NO: 62 and a reverseprimer comprising or consisting essentially of SEQ ID NO: 63.

The amplified Acinetobacter baumannii, Pseudomonas aeruginosa,Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis,Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystisjirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasmaparvum ure, Ureaplasma spp. Bartonella spp., Streptococcus agalactiae,or Neisseria meningitidis nucleic acid can be detected in real-time, forexample by real-time PCR, in order to determine the presence and/or theamount of a pathogen nucleic acid in a sample. In this manner, anamplified nucleic acid sequence can be detected using a probe specificfor the product amplified from the target sequence of interest. Suitableprobes for real-time PCR include those described herein, such as a probehaving a nucleic acid sequence at least 90% identical to SEQ ID NO: 14,17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 56, 60, or 64. Inparticular examples of the disclosed methods, multiplex real-time PCR isutilized to detect one or more (for example, at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15) of an Acinetobacter baumannii,Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii,Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus,Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum,Ureaplasma parvum, Ureaplasma spp. Bartonella spp., Streptococcusagalactiae, or Neisseria meningitidis nucleic acid present in thesample. In other examples of the disclosed methods, simultaneousmultipathogen detection (such as multiple singleplex real-time PCRreactions, for example on a single array or card) is utilized to detectone or more (for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15) of an Acinetobacter baumannii, Pseudomonas aeruginosa,Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis,Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystisjirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasmaparvum, Ureaplasma spp. Bartonella spp., Streptococcus agalactiae, orNeisseria meningitidis nucleic acid present in the sample.

Real-time PCR monitors the fluorescence emitted during the reaction asan indicator of amplicon production during each PCR cycle, as opposed toendpoint detection. The real-time progress of the reaction can be viewedin some systems. Typically, real-time PCR uses the detection of afluorescent reporter. Typically, the fluorescent reporter's signalincreases in direct proportion to the amount of PCR product in areaction. By recording the amount of fluorescence emission at eachcycle, it is possible to monitor the PCR reaction during exponentialphase where the first significant increase in the amount of PCR productcorrelates to the initial amount of target template. The higher thestarting copy number of the nucleic acid target, the sooner asignificant increase in fluorescence is observed.

In one embodiment, the fluorescently-labeled probes (such as probesdisclosed herein) rely upon fluorescence resonance energy transfer(FRET), or in a change in the fluorescence emission wavelength of asample, as a method to detect hybridization of a DNA probe to theamplified target nucleic acid in real-time. For example, FRET thatoccurs between fluorogenic labels on different probes (for example,using HybProbes) or between a donor fluorophore and an acceptor orquencher fluorophore on the same probe (for example, using a molecularbeacon or a TAQMAN® probe) can identify a probe that specificallyhybridizes to the nucleic acid of interest and in this way, using anAcinetobacter baumannii oxa-51 probe, a Pseudomonas aeruginosa gyrBprobe, a Klebsiella pneumoniae nifA probe or a Klebsiella pneumoniaediguanylate cyclase probe, a Toxoplasma gondii ssrRNA probe, a Moraxellacatarrhalis purH probe, an Escherichia coli/Shigella uidA probe, aStaphylococcus aureus gsf probe, a Pneumocystis jirovecii dhps probe, aChlamydia trachomatis tmRNA probe, a Ureaplasma urealyticum mba probe, aUreaplasma parvum ure probe, a Ureaplasma spp. ure probe, a Bartonellaspp. ssrA probe, a Streptococcus agalactiae cfb probe, or a Neisseriameningitidis sodC probe can detect the presence and/or amount of therespective pathogen in a sample.

In some embodiments, the fluorescently-labeled DNA probes used toidentify amplification products have spectrally distinct emissionwavelengths, thus allowing them to be distinguished within the samereaction tube, for example in multiplex PCR, such as a multiplexreal-time PCR. In some embodiments, the probes and primers disclosedherein are used in multiplex real-time PCR. For example, multiplex PCRpermits the simultaneous detection of one or more (for example, at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the amplificationproducts of Acinetobacter baumannii oxa-51 nucleic acid, a Pseudomonasaeruginosa gyrB nucleic acid, a Klebsiella pneumoniae nifA nucleic acid,a Klebsiella pneumoniae diguanylate cyclase nucleic acid, a Toxoplasmagondii ssrRNA nucleic acid, a Moraxella catarrhalis purH nucleic acid,an Escherichia coli/Shigella uidA nucleic acid, a Staphylococcus aureusgsf nucleic acid, a Pneumocystis jirovecii dhps nucleic acid, aChlamydia trachomatis tmRNA nucleic acid, a Ureaplasma urealyticum mbanucleic acid, a Ureaplasma parvum ure nucleic acid, a Ureaplasma spp.ure nucleic acid, a Bartonella spp. ssrA nucleic acid, a Streptococcusagalactiae cfb nucleic acid, and a Neisseria meningitidis sodC nucleicacid. Using the disclosed primers and probes, any combination ofAcinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum ure, Ureaplasma spp.,Bartonella spp. Streptococcus agalactiae, and Neisseria meningitidisnucleic acids can be detected. In some examples, the multiplex reactionmay include one or more of the primers and probes disclosed herein andprimers and probes for detection of additional pathogens.

In other examples, the probes and primers disclosed herein are used in abead-based multiplex assay (see, e.g., U.S. Pat. No. 6,939,720). Forexample, probes specific for each pathogen (such as the probes disclosedherein) are attached to different fluorescently labeled beads and arehybridized to amplified DNA from the sample. The probes will onlysignificantly hybridize if the particular pathogen is present in thesample. The hybridized beads are then captured, for example with abiotinylated detector molecule, and the relative fluorescence of thebeads for each label is measured.

In another embodiment, a melting curve analysis of the amplified targetnucleic acid can be performed subsequent to the amplification process.The T_(m) of a nucleic acid sequence depends on the length of thesequence and its G/C content. Thus, the identification of the T_(m) fora nucleic acid sequence can be used to identify the amplified nucleicacid, for example by using double-stranded DNA binding dye chemistry,which quantitates the amplicon production by the use of a non-sequencespecific fluorescent intercalating agent (such as SYBR® Green orethidium bromide). SYBR® Green is a fluorogenic minor groove binding dyethat exhibits little fluorescence when in solution but emits a strongfluorescent signal upon binding to double-stranded DNA. Typically, SYBR®Green is used in singleplex reactions, however when coupled with meltingpoint analysis, it can be used for multiplex reactions.

Any type of thermal cycler apparatus can be used for the amplificationof pathogen or control nucleic acids and/or the determination ofhybridization. Examples of suitable apparatuses include PTC-100® PeltierThermal Cycler (MJ Research, Inc.; San Francisco, Calif.), ROBOCYCLER®40 Temperature Cycler (Agilent/Stratagene; Santa Clara, Calif.), orGENEAMP® PCR System 9700 (Applied Biosystems; Foster City, Calif.). Forreal-time PCR, any type of real-time thermocycler apparatus can be used.For example, iCYCLER iQ™ or CFX96™ real-time detection systems (Bio-Rad,Hercules, Calif.), LightCycler® systems (Roche, Mannheim, Germany), ABI™systems such as the 7000, 7300, 7500, 7700, or 7900 systems or the VIIA™7 real-time PCR system (Applied Biosystems; Foster City, Calif.),MX4000™, MX3000™ or MX3005™ qPCR systems (Agilent/Stratagene; SantaClara, Calif.), DNA Engine OPTICON® Continuous Fluorescence DetectionSystem (Bio-Rad, Hercules, Calif.), ROTOR-GENE® Q real-time cycler(Qiagen, Valencia, Calif.), or SMARTCYCLER® system (Cepheid, Sunnyvale,Calif.) can be used to amplify nucleic acid sequences in real-time. Insome embodiments, real-time PCR is performed using a TAQMAN® arrayformat, for example, a microfluidic card in which each well ispre-loaded with primers and probes for a particular target. The reactionis initiated by adding a sample including nucleic acids and assayreagents (such as a PCR master mix) and running the reactions in areal-time thermocycler apparatus.

In some embodiments, a microfluidic card includes at least one wellcontaining primers and probes for at least one of Acinetobacterbaumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasmagondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcusaureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasmaurealyticum, Ureaplasma parvum ure, Ureaplasma spp. ure, Bartonella spp.ssrA nucleic acid, Streptococcus agalactiae, Neisseria meningitidis, orany combination or two or more thereof (such as 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 thereof). In one non-limiting example, thecard includes at least one well containing Acinetobacter baumanniiprimers and probe (e.g., SEQ ID NOs: 12-14), at least one wellcontaining Pseudomonas aeruginosa primers and probe (e.g., SEQ ID NOs:15-17), at least one well containing Klebsiella pneumoniae primers andprobe (e.g., SEQ ID NOs: 18-20 or SEQ ID NOs: 58-60), at least one wellcontaining Toxoplasma gondii primers and probe (e.g., SEQ ID NOs:21-23), at least one well containing Moraxella catarrhalis primers andprobe (e.g., SEQ ID NOs: 24-26), at least one well containing E.coli/Shigella primers and probe (e.g., SEQ ID NOs: 27-29), at least onewell containing Staphylococcus aureus primers and probe (e.g., SEQ IDNOs: 30-32), at least one well containing Pneumocystis jirovecii primersand probe (e.g., SEQ ID NOs: 33-35), at least one well containingChlamydia trachomatis primers and probe (e.g., SEQ ID NOs: 36-38), atleast one well containing Ureaplasma urealyticum primers and probe(e.g., SEQ ID NOs: 39-41), at least one well containing Ureaplasmaparvum primers and probe (e.g., SEQ ID NOs: 42-44), at least one wellcontaining Ureaplasma spp. primers and probe (e.g., SEQ ID NOs: 45-47),at least one well containing Bartonella spp. primers and probe (e.g.,SEQ ID NOs: 48-50 and/or SEQ ID NOs: 49-51), at least one wellcontaining Streptococcus agalactiae primers and probe (e.g., SEQ ID NOs:54-56), and at least one well containing Neisseria meningitidis primersand probe (e.g., SEQ ID NOs: 62-64).

In another non-limiting example, the card includes at least one wellcontaining Chlamydia trachomatis primers and probe (e.g., SEQ ID NOs:36-38) and at least one well containing Ureaplasma spp. primers andprobe (e.g., SEQ ID NOs: 45-47). In yet another non-limiting example,the card includes at least one well containing Staphylococcus aureusprimers and probe (e.g., SEQ ID NOs: 30-32), at least one wellcontaining Pseudomonas aeruginosa primers and probe (e.g., SEQ ID NOs:15-17), at least one well containing E. coli/Shigella primers and probe(e.g., SEQ ID NOs: 27-29), at least one well containing Klebsiellapneumoniae primers and probe (e.g., SEQ ID NOs: 18-20), at least onewell containing Acinetobacter baumannii primers and probe (e.g., SEQ IDNOs: 12-14), at least one well containing Toxoplasma gondii primers andprobe (e.g., SEQ ID NOs: 21-23), at least one well containing Ureaplasmaspp. primers and probe (e.g., SEQ ID NOs: 45-47), at least one wellcontaining Chlamydia trachomatis primers and probe (e.g., SEQ ID NOs:36-38), and at least one well containing Streptococcus agalactiaeprimers and probe (e.g., SEQ ID NOs: 54-56). In some examples, this cardmay also include at least one well containing Neisseria meningitidisprimers and probe (e.g., SEQ ID NOs: 62-64) and/or at least one wellcontaining Salmonella spp. primers and probe (e.g., SEQ ID NOs: 65-67).The card may include additional primers and probes in additional wells,such as positive control primers and probes, or primers and probes foradditional pathogens or other nucleic acids of interest. In someexamples, a card may additionally include wells containing primers andprobes for one or more of Mycoplasma pneumoniae, Chlamydophilapneumoniae, Bordetella pertussis, adenovirus, influenza virus (A or B),parainfluenza virus (type 1, 2, or 3), respiratory syncytial virus,parechovirus, enterovirus, human metapneumovirus, rubella, Streptococcuspneumoniae, Streptococcus pyogenes, rhinovirus, Group B Streptococcus,Herpes simplex virus (1 or 2), pan-Haemophilus influenzae,pan-Salmonella, Neisseria meningitidis, cytomegalovirus, or anycombination of two or more thereof. Additional combinations of assayscan be selected and included on a TAC, as will be understood by one ofordinary skill in the art.

In some embodiments, the probe is detectably labeled, either with anisotopic or non-isotopic label; in alternative embodiments, the targetnucleic acid is labeled. Non-isotopic labels can, for instance, comprisea fluorescent or luminescent molecule, or an enzyme, co-factor, enzymesubstrate, or hapten. The probe is incubated with a single-stranded ordouble-stranded preparation of RNA, DNA, or a mixture of both, andhybridization is determined. In some examples, the hybridization resultsin a detectable change in signal such as in increase or decrease insignal, for example from the labeled probe. Thus, detectinghybridization comprises detecting a change in signal from the labeledprobe during or after hybridization relative to signal from the labelbefore hybridization.

In some examples, the disclosed methods can predict with a sensitivityof at least 90% and a specificity of at least 90% for presence of anAcinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, and/or Neisseria meningitidis nucleicacid, such as a sensitivity of at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or even 100% and a specificity of at least of at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%.

IV. Probes and Primers

Probes and primers suitable for use in the disclosed methods aredescribed herein. Such probes and primers include nucleic acid moleculescapable of hybridizing to the disclosed nucleic acid molecules, such asSEQ ID NOs: 1-11 or 52.

A. Probes

Probes capable of hybridizing to and detecting the presence ofAcinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acidmolecules are disclosed. In some embodiments, the disclosed probes arebetween 10 and 40 nucleotides in length, such as 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 29, 30, 31, 32, 32,34, 35, 36, 37, 38, 39, or 40 nucleotides in length and are capable ofhybridizing to the disclosed nucleic acid molecules. In some examples,the probes are at least 10, 15, 20, 25, 30, 35, or 40 nucleotides inlength. In other examples, the probes may be no more than 10, 15, 20,25, 30, 35, or 40 nucleotides in length. The disclosed probes may alsoinclude a 3′ C6 CpG in some examples.

In several embodiments, a probe is capable of hybridizing under high orvery high stringency conditions to an Acinetobacter baumannii nucleicacid sequence set forth as SEQ ID NO: 1. In some examples, a probecapable of hybridizing to an Acinetobacter baumannii oxa-51 nucleicmolecule includes a nucleic acid sequence that is at least 90%identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or even 100% identical, to the nucleotide sequence set forth asTGACTGCTAATCCAAATCACAGCGCTTCA (SEQ ID NO: 14). In several embodiments, aprobe capable of hybridizing to an Acinetobacter baumannii oxa-51nucleic acid molecule consists essentially of, or consists of, a nucleicacid sequence set forth as SEQ ID NO: 14.

In some embodiments, a probe is capable of hybridizing under high orvery high stringency conditions to a Chlamydia trachomatis nucleic acidsequence set forth as SEQ ID NO: 2. In some examples, a probe capable ofhybridizing to a Chlamydia trachomatis tmRNA nucleic molecule includes anucleic acid sequence that is at least 90% identical, such as at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, tothe nucleotide sequence set forth as ATGCGGAGGGCGTTGGCTGG (SEQ ID NO:38). In several embodiments, a probe capable of hybridizing to aChlamydia trachomatis tmRNA nucleic acid molecule consists essentiallyof, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 38.

In other embodiments, a probe is capable of hybridizing under high orvery high stringency conditions to an Escherichia coli and/or Shigellaspp. nucleic acid sequence set forth as SEQ ID NO: 3. In some examples,a probe capable of hybridizing to an Escherichia coli and/or Shigellaspp. nucleic molecule includes a nucleic acid sequence that is at least90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or even 100% identical, to the nucleotide sequence set forth asTACGGCGTGACATCGGCTTCAAATG (SEQ ID NO: 29). In several embodiments, aprobe capable of hybridizing to an Escherichia coli uidA nucleic acidmolecule consists essentially of, or consists of, a nucleic acidsequence set forth as SEQ ID NO: 29. In particular embodiments, theprobe capable of hybridizing to an Escherichia coli uidA nucleic acidmolecule is capable of hybridizing to a uidA nucleic acid molecule fromany an Escherichia coli and/or Shigella species or serogroup.

In additional embodiments, a probe is capable of hybridizing under highor very high stringency conditions to a Klebsiella pneumoniae nucleicacid sequence set forth as SEQ ID NO: 4. In some examples, a probecapable of hybridizing to a Klebsiella pneumoniae nifA nucleic moleculeincludes a nucleic acid sequence that is at least 90% identical, such asat least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%identical, to the nucleotide sequence set forth asACGCTGAGCACCTCCTGCAACGT (SEQ ID NO: 20). In several embodiments, a probecapable of hybridizing to a Klebsiella pneumoniae nifA nucleic acidmolecule consists essentially of, or consists of, a nucleic acidsequence set forth as SEQ ID NO: 20. In other embodiments, a probe iscapable of hybridizing under high or very high stringency conditions toa Klebsiella pneumoniae nucleic acid sequence set forth as SEQ ID NO:57. In some examples, a probe capable of hybridizing to a Klebsiellapneumoniae diguanylate cyclase nucleic molecule includes a nucleic acidsequence that is at least 90% identical, such as at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotidesequence set forth as CCACCACGCTCATCTGTTTCGCC (SEQ ID NO: 60). Inseveral embodiments, a probe capable of hybridizing to a Klebsiellapneumoniae diguanylate cyclase nucleic acid molecule consistsessentially of, or consists of, a nucleic acid sequence set forth as SEQID NO: 60.

In further embodiments, a probe is capable of hybridizing under high orvery high stringency conditions to a Moraxella catarrhalis nucleic acidsequence set forth as SEQ ID NO: 5. In some examples, a probe capable ofhybridizing to a Moraxella catarrhalis purH nucleic molecule includes anucleic acid sequence that is at least 90% identical, such as at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, tothe nucleotide sequence set forth as CACAGCGGGCAGCTCAATTTGACCTA (SEQ IDNO: 26). In several embodiments, a probe capable of hybridizing to aMoraxella catarrhalis purH nucleic acid molecule consists essentiallyof, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 26.

In still further embodiments, a probe is capable of hybridizing underhigh or very high stringency conditions to a Pneumocystis jiroveciinucleic acid sequence set forth as SEQ ID NO: 6. In some examples, aprobe capable of hybridizing to a Pneumocystis jirovecii dhps nucleicmolecule includes a nucleic acid sequence that is at least 90%identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or even 100% identical, to the nucleotide sequence set forth asACAGGGTGTCTTACAGGTGATGTTATGCCAAAAG (SEQ ID NO: 35). In severalembodiments, a probe capable of hybridizing to a Pneumocystis jiroveciidhps nucleic acid molecule consists essentially of, or consists of, anucleic acid sequence set forth as SEQ ID NO: 35. In one example, aprobe capable of hybridizing to a Pneumocystis jirovecii dhps nucleicacid molecule includes or consists of a nucleic acid sequence set forthas ACAGGGTGTCT“T”ACAGGTGATGTTATGCCAAAAG (SEQ ID NO: 35), where “T” isBHQ1.

In additional embodiments, a probe is capable of hybridizing under highor very high stringency conditions to a Pseudomonas aeruginosa nucleicacid sequence set forth as SEQ ID NO: 7. In some examples, a probecapable of hybridizing to a Pseudomonas aeruginosa gyrB nucleic moleculeincludes a nucleic acid sequence that is at least 90% identical, such asat least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%identical, to the nucleotide sequence set forth asTCCGTCGCCACAACAAGGTCTGGGAA (SEQ ID NO: 17). In several embodiments, aprobe capable of hybridizing to a Pseudomonas aeruginosa gyrB nucleicacid molecule consists essentially of, or consists of, a nucleic acidsequence set forth as SEQ ID NO: 17.

In other embodiments, a probe is capable of hybridizing under high orvery high stringency conditions to a Staphylococcus aureus nucleic acidsequence set forth as SEQ ID NO: 8. In some examples, a probe capable ofhybridizing to a Staphylococcus aureus gsf nucleic molecule includes anucleic acid sequence that is at least 90% identical, such as at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, tothe nucleotide sequence set forth as TTCCATATGACCACCACGAGTCTTAGCACC (SEQID NO: 32). In several embodiments, a probe capable of hybridizing to aStaphylococcus aureus gsf nucleic acid molecule consists essentially of,or consists of, a nucleic acid sequence set forth as SEQ ID NO: 32.

In some embodiments, a probe is capable of hybridizing under high orvery high stringency conditions to a Toxoplasma gondii nucleic acidsequence set forth as SEQ ID NO: 9. In some examples, a probe capable ofhybridizing to a Toxoplasma gondii ssrRNA nucleic molecule includes anucleic acid sequence that is at least 90% identical, such as at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, tothe nucleotide sequence set forth as ATCGCGTTGACTTCGGTCTGCGAC (SEQ IDNO: 23). In several embodiments, a probe capable of hybridizing to aToxoplasma gondii ssrRNA nucleic acid molecule consists essentially of,or consists of, a nucleic acid sequence set forth as SEQ ID NO: 23.

In some embodiments, a probe is capable of hybridizing under high orvery high stringency conditions to a Ureaplasma parvum nucleic acidsequence set forth as SEQ ID NO: 10. In some examples, a probe capableof hybridizing to a Ureaplasma parvum ure nucleic molecule includes anucleic acid sequence that is at least 90% identical, such as at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, tothe nucleotide sequence set forth as TCAGTGAACGTGAGTATCTAAACCACCAGC (SEQID NO: 44). In several embodiments, a probe capable of hybridizing to aUreaplasma parvum ure nucleic acid molecule consists essentially of, orconsists of, a nucleic acid sequence set forth as SEQ ID NO: 44. In someexamples, a probe capable of hybridizing to a Ureaplasma parvum urenucleic acid molecule includes or consists of a nucleic acid sequenceset forth as TCAGTGAACG“T”GAGTATCTAAACCACCAGC (SEQ ID NO: 44), where “T”is BHQ1.

In additional embodiments, a probe is capable of hybridizing under highor very high stringency conditions to a Ureaplasma urealyticum nucleicacid sequence set forth as SEQ ID NO: 11. In some examples, a probecapable of hybridizing to a Ureaplasma urealyticum mba nucleic moleculeincludes a nucleic acid sequence that is at least 90% identical, such asat least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%identical, to the nucleotide sequence set forth asCACAGCAACTACCCCTGCTCCCACTAA (SEQ ID NO: 41). In several embodiments, aprobe capable of hybridizing to a Ureaplasma urealyticum mba nucleicacid molecule consists essentially of, or consists of, a nucleic acidsequence set forth as SEQ ID NO: 41.

In several embodiments, a probe capable of hybridizing to a Ureaplasmaspp. ure nucleic molecule contains a nucleic acid sequence that is atleast 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or even 100% identical, to the nucleotide sequence set forth asCCACCAGCAATAACAGTTGTAATACCACCATC (SEQ ID NO: 47). In severalembodiments, a probe capable of hybridizing to a Ureaplasma spp. urenucleic acid molecule consists essentially of, or consists of, a nucleicacid sequence set forth as SEQ ID NO: 47. In several examples, a probecapable of hybridizing to a Ureaplasma spp. ure nucleic acid moleculeincludes or consists of a nucleic acid sequence set forth asCCACCAGCAA“T”AACAGTTGTAATACCACCATC (SEQ ID NO: 47), where “T” is BHQ1.In particular embodiments, the probe capable of hybridizing to aUreaplasma spp. ure nucleic acid molecule is capable of hybridizing to aure nucleic acid molecule from any Ureaplasma species or serogroup (forexample, Ureaplasma parvum or Ureaplasma urealyticum).

In additional embodiments, a probe is capable of hybridizing under highor very high stringency conditions to a Bartonella spp. nucleic acidsequence set forth as SEQ ID NO: 52. In several embodiments, a probecapable of hybridizing to a Bartonella spp. ssrA nucleic moleculecontains a nucleic acid sequence that is at least 90% identical, such asat least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%identical, to the nucleotide sequence set forth as ACCCCGCTTAAACCTGCGACG(SEQ ID NO: 50). In several embodiments, a probe capable of hybridizingto a Bartonella spp. ssrA nucleic acid molecule consists essentially of,or consists of, a nucleic acid sequence set forth as SEQ ID NO: 50. Inparticular embodiments, the probe capable of hybridizing to a Bartonellaspp. ssrA nucleic acid molecule is capable of hybridizing to a ssrAnucleic acid molecule from any Bartonella species or serogroup (forexample, B. alsatica, B. bacilliformis, B. birtlesii, B. bovis, Bcapreoli, B. chomelii, B. clarridgeiae, B. doshiae, B. elizabethae, B.henselae, B. grahamii, B. japonica, B. koehlerae, B. melophagi, B.phoceensis, B. quintana, B. rochalimae, B. schoenbuchensis, B.silvatica, B. tamiae, B. taylorii, B. tribocorum, B. vinsonii, and/or B.washoensis).

In some embodiments, a probe is capable of hybridizing under high orvery high stringency conditions to a Streptococcus agalactiae nucleicacid sequence set forth as SEQ ID NO: 53. In some examples, a probecapable of hybridizing to a Streptococcus agalactiae cfb nucleicmolecule includes a nucleic acid sequence that is at least 90%identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or even 100% identical, to the nucleotide sequence set forth asAGACTTCATTGCGTGCCAACCCTGAGAC (SEQ ID NO: 56). In several embodiments, aprobe capable of hybridizing to a Streptococcus agalactiae cfb nucleicacid molecule consists essentially of, or consists of, a nucleic acidsequence set forth as SEQ ID NO: 56.

In further embodiments, a probe is capable of hybridizing under high orvery high stringency conditions to a Neisseria meningitidis nucleic acidsequence set forth as SEQ ID NO: 61. In some examples, a probe capableof hybridizing to a Neisseria meningitidis sodC nucleic moleculeincludes a nucleic acid sequence that is at least 90% identical, such asat least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%identical, to the nucleotide sequence set forth as CGCAGGCGGTCACTGGGATC(SEQ ID NO: 64). In several embodiments, a probe capable of hybridizingto a Neisseria meningitidis sodC nucleic acid molecule consistsessentially of, or consists of, a nucleic acid sequence set forth as SEQID NO: 64.

In some examples, the probe is labeled with one or more fluorophores.Examples of suitable fluorophore labels are provided above. In someexamples, the fluorophore is a donor fluorophore. In particular,non-limiting examples, the probes disclosed herein are labeled with FAM,although one of ordinary skill in the art can select other fluorophorelabels for use in the disclosed methods. In other examples, thefluorophore is an accepter fluorophore, such as a fluorescence quencher.In some examples, the probe includes both a donor fluorophore and anaccepter or quencher fluorophore, for example a donor fluorophore suchas a FAM and an acceptor fluorophore such as a BLACK HOLE® quencher(such as BHQ1, BHQ2, or BHQ3) or TAMRA. Appropriate donor/acceptorfluorophore pairs can be selected using routine methods. In one example,the donor emission wavelength is one that can significantly excite theacceptor, thereby generating a detectable emission from the acceptor. Insome examples, the probe is modified at the 3′-end to prevent extensionof the probe by a polymerase.

In some examples, the acceptor fluorophore (such as a fluorescencequencher) is attached to the 3′ end of the probe and the donorfluorophore is attached to a 5′ end of the probe. In other examples, theacceptor fluorophore (such as a fluorescence quencher) is attached tothe 5′ end of the probe and the donor fluorophore is attached to a 3′end of the probe. In another particular example, the acceptorfluorophore (such as a fluorescence quencher) is attached to a modifiednucleotide (such as a T) and the donor fluorophore is attached to a 5′end of the probe. In some examples, the donor fluorophore is FAM and theacceptor fluorophore is BHQ1. In particular embodiments, the probesdisclosed herein include a donor fluorophore attached to the 5′ end andan acceptor fluorophore attached to the 3′ end.

B. Primers

Primers capable of hybridizing to and directing the amplification ofAcinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acidmolecules are also disclosed. The primers disclosed herein are between10 to 40 nucleotides in length, such as 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, or even 40 nucleotides in length. In some examples, theprimers are at least 10, 15, 20, 25, 30, 35, or 40 nucleotides inlength. In other examples, the primers may be no more than 10, 15, 20,25, 30, 35, or 40 nucleotides in length.

In several embodiments, a primer is capable of hybridizing to anddirecting the amplification of an Acinetobacter baumannii oxa-51 nucleicacid molecule (such as SEQ ID NO: 1) and includes a nucleic acidsequence that is at least 90% identical, such as at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleicacid sequence set forth as TATTTTTATTTCAGCCTGCTCACCTT (SEQ ID NO: 12) orAAATACTTCTGTGGTGGTTGCCTTA (SEQ ID NO: 13). In several embodiments, aprimer capable of hybridizing to and directing the amplification of anAcinetobacter baumannii oxa-51 nucleic acid molecule consistsessentially of, or consists of a nucleic acid sequence set forth as SEQID NO: 12 or SEQ ID NO: 13.

In some embodiments, a primer is capable of hybridizing to and directingthe amplification of a Chlamydia trachomatis tmRNA nucleic acid molecule(such as SEQ ID NO: 2) and includes a nucleic acid sequence that is atleast 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or even 100% identical, to the nucleic acid sequence set forthas GGTGTAAAGGTTTCGACTTAGAA (SEQ ID NO: 36) or CGAACACCGGGTCACC (SEQ IDNO: 37). In several embodiments, a primer capable of hybridizing to anddirecting the amplification of a Chlamydia trachomatis tmRNA nucleicacid molecule consists essentially of, or consists of a nucleic acidsequence set forth as SEQ ID NO: 36 or SEQ ID NO: 37.

In other embodiments, a primer is capable of hybridizing to anddirecting the amplification of an Escherichia coli and/or Shigella spp.nucleic acid molecule (such as SEQ ID NO: 3) and includes a nucleic acidsequence that is at least 90% identical, such as at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleicacid sequence set forth as GAGCATCAGGGTGGCTATACG (SEQ ID NO: 27) orATAGTCTGCCAGTTCAGTTC (SEQ ID NO: 28). In several embodiments, a primercapable of hybridizing to and directing the amplification of anEscherichia coli and/or Shigella spp. nucleic acid molecule consistsessentially of, or consists of a nucleic acid sequence set forth as SEQID NO: 27 or SEQ ID NO: 28.

In additional embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Klebsiella pneumoniae nifA nucleic acidmolecule (such as SEQ ID NO: 4) and includes a nucleic acid sequencethat is at least 90% identical, such as at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acidsequence set forth as TGCTGCATAAAGGCATCGTT (SEQ ID NO: 18) orCCACCGAGGCCAGCAA (SEQ ID NO: 19). In several embodiments, a primercapable of hybridizing to and directing the amplification of aKlebsiella pneumoniae nifA nucleic acid molecule consists essentiallyof, or consists of a nucleic acid sequence set forth as SEQ ID NO: 18 orSEQ ID NO: 19. In further embodiments, a primer is capable ofhybridizing to and directing the amplification of a Klebsiellapneumoniae diguanylate cyclase nucleic acid molecule (such as SEQ ID NO:57) and includes a nucleic acid sequence that is at least 90% identical,such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even100% identical, to the nucleic acid sequence set forth asTGCAGATAATTCACGCCCAG (SEQ ID NO: 58) or ACCCGCTGGACGCCAT (SEQ ID NO:59). In several embodiments, a primer capable of hybridizing to anddirecting the amplification of a Klebsiella pneumoniae diguanylatecyclase nucleic acid molecule consists essentially of, or consists of anucleic acid sequence set forth as SEQ ID NO: 58 or SEQ ID NO: 59.

In further embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Moraxella catarrhalis purH nucleic acidmolecule (such as SEQ ID NO: 5) and includes a nucleic acid sequencethat is at least 90% identical, such as at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acidsequence set forth as GGTGAGTTGCCACAGC (SEQ ID NO: 24) orAGTAGACCGCCATTGACTC (SEQ ID NO: 25). In several embodiments, a primercapable of hybridizing to and directing the amplification of a Moraxellacatarrhalis purH nucleic acid molecule consists essentially of, orconsists of a nucleic acid sequence set forth as SEQ ID NO: 24 or SEQ IDNO: 25.

In still further embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Pneumocystis jirovecii dhps nucleicacid molecule (such as SEQ ID NO: 6) and includes a nucleic acidsequence that is at least 90% identical, such as at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleicacid sequence set forth as TAATGGTTTGCCTTGGTTGCTT (SEQ ID NO: 33) orCACAGCCTCCTAAAACAGAT (SEQ ID NO: 34). In several embodiments, a primercapable of hybridizing to and directing the amplification of aPneumocystis jirovecii dhps nucleic acid molecule consists essentiallyof, or consists of a nucleic acid sequence set forth as SEQ ID NO: 33 orSEQ ID NO: 34.

In other embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Pseudomonas aeruginosa gyrB nucleicacid molecule (such as SEQ ID NO: 7) and includes a nucleic acidsequence that is at least 90% identical, such as at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleicacid sequence set forth as GTCTCGGTGGTGAACG (SEQ ID NO: 15) orTGGATGTTGCTGAAGGTCTC (SEQ ID NO: 16). In several embodiments, a primercapable of hybridizing to and directing the amplification of aPseudomonas aeruginosa gyrB nucleic acid molecule consists essentiallyof, or consists of a nucleic acid sequence set forth as SEQ ID NO: 15 orSEQ ID NO: 16.

In additional embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Staphylococcus aureus gsf nucleic acidmolecule (such as SEQ ID NO: 8) and includes a nucleic acid sequencethat is at least 90% identical, such as at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acidsequence set forth as CGGGTTAGGTGAATTGATTGTTTTAT (SEQ ID NO: 30) orCGCATTTGAGCTGAAGTTG (SEQ ID NO: 31). In several embodiments, a primercapable of hybridizing to and directing the amplification of aStaphylococcus aureus gsf nucleic acid molecule consists essentially of,or consists of a nucleic acid sequence set forth as SEQ ID NO: 30 or SEQID NO: 31.

In other embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Toxoplasma gondii ssrRNA nucleic acidmolecule (such as SEQ ID NO: 9) and includes a nucleic acid sequencethat is at least 90% identical, such as at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acidsequence set forth as GGTGGTCCTCAGGTGAT (SEQ ID NO: 21) orCCACGGTAGTCCAATACAGTA (SEQ ID NO: 22). In several embodiments, a primercapable of hybridizing to and directing the amplification of aToxoplasma gondii ssrRNA nucleic acid molecule consists essentially of,or consists of a nucleic acid sequence set forth as SEQ ID NO: 21 or SEQID NO: 22.

In further embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Ureaplasma parvum ure nucleic acidmolecule (such as SEQ ID NO: 10) and includes a nucleic acid sequencethat is at least 90% identical, such as at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acidsequence set forth as ACAGATAATGTTGATATGATTGTGGGTAT (SEQ ID NO: 42) orCTAATGCAACAGGAACTATTTCTG (SEQ ID NO: 43). In several embodiments, aprimer capable of hybridizing to and directing the amplification of aUreaplasma parvum ure nucleic acid molecule consists essentially of, orconsists of a nucleic acid sequence set forth as SEQ ID NO: 42 or SEQ IDNO: 43.

In still further embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Ureaplasma urealyticum mba nucleic acidmolecule (such as SEQ ID NO: 11) and includes a nucleic acid sequencethat is at least 90% identical, such as at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acidsequence set forth as ATTTCATATTTAGTTTATTAGGAGATCGTTAT (SEQ ID NO: 39)or AGATTTAACATTTGAGCTAGAACAT (SEQ ID NO: 40). In several embodiments, aprimer capable of hybridizing to and directing the amplification of aUreaplasma urealyticum mba nucleic acid molecule consists essentiallyof, or consists of a nucleic acid sequence set forth as SEQ ID NO: 39 orSEQ ID NO: 40.

In additional embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Ureaplasma spp. ure nucleic acidmolecule (such as SEQ ID NO: 10) and includes a nucleic acid sequencethat is at least 90% identical, such as at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acidsequence set forth as GGTTTAGATACTCACGTTCACTGA (SEQ ID NO: 45) orGCTTTTGTACCATCATTCATACCTGT (SEQ ID NO: 46). In several embodiments, aprimer capable of hybridizing to and directing the amplification of aUreaplasma spp. ure nucleic acid molecule consists essentially of, orconsists of a nucleic acid sequence set forth as SEQ ID NO: 45 or SEQ IDNO: 46.

In additional embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Bartonella spp. ssrA nucleic acidmolecule (such as SEQ ID NO: 52) and includes a nucleic acid sequencethat is at least 90% identical, such as at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acidsequence set forth as GCTATGGTAATAAATGGACAATGAAATAA (SEQ ID NO: 48),GCTTCTGTTGCCAGGTG (SEQ ID NO: 49), or CTAAATGAGTAGTTGCAAATGACAAC (SEQ IDNO: 51). In several embodiments, a primer capable of hybridizing to anddirecting the amplification of a Bartonella spp. ssrA nucleic acidmolecule consists essentially of, or consists of a nucleic acid sequenceset forth as SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 51. Inparticular embodiments, the primer capable of hybridizing to anddirecting amplification of a Bartonella spp. ssrA nucleic acid moleculeis capable of hybridizing to a ssrA nucleic acid molecule from anyBartonella species or serogroup (for example, B. alsatica, B.bacilliformis, B. birtlesii, B. bovis, B capreoli, B. chomelii, B.clarridgeiae, B. doshiae, B. elizabethae, B. henselae, B. grahamii, B.japonica, B. koehlerae, B. melophagi, B. phoceensis, B. quintana, B.rochalimae, B. schoenbuchensis, B. silvatica, B. tamiae, B. taylorii, B.tribocorum, B. vinsonii, and/or B. washoensis).

In additional embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Streptococcus agalactiae cfb nucleicacid molecule (such as SEQ ID NO: 53) and includes a nucleic acidsequence that is at least 90% identical, such as at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleicacid sequence set forth as GGGAACAGATTATGAAAAACCG (SEQ ID NO: 54) orAAGGCTTCTACACGACTACCAA (SEQ ID NO: 55). In several embodiments, a primercapable of hybridizing to and directing the amplification of aStreptococcus agalactiae cfb nucleic acid molecule consists essentiallyof, or consists of a nucleic acid sequence set forth as SEQ ID NO: 54 orSEQ ID NO: 55.

In additional embodiments, a primer is capable of hybridizing to anddirecting the amplification of a Neisseria meningitidis sodC nucleicacid molecule (such as SEQ ID NO: 61) and includes a nucleic acidsequence that is at least 90% identical, such as at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleicacid sequence set forth as CTGTGAGCCAAAAGAAAAAGAAG (SEQ ID NO: 62) orGATTTGTTGCTGTGCCATCAT (SEQ ID NO: 63). In several embodiments, a primercapable of hybridizing to and directing the amplification of a Neisseriameningitidis sodC nucleic acid molecule consists essentially of, orconsists of a nucleic acid sequence set forth as SEQ ID NO: 62 or SEQ IDNO: 63.

In certain embodiments, the primers are utilized or provided as a set ofprimers, such as a pair of primers, capable of hybridizing to andamplifying a disclosed nucleic acid, such as one of SEQ ID NOs: 1-11,52-53, 57, or 60. In some examples, the set of primers includes a pairof primers including SEQ ID NOs: 12 and 13, a pair of primers includingSEQ ID NOs: 15 and 16, a pair of primers including SEQ ID NOs: 18 and19, a pair of primers including SEQ ID NOs: 21 and 22, a pair of primersincluding SEQ ID NOs: 24 and 25, a pair of primers including SEQ ID NOs:27 and 28, a pair of primers including SEQ ID NOs: 30 and 31, a pair ofprimers including SEQ ID NOs: 33 and 34, a pair of primers including SEQID NOs: 36 and 37, a pair of primers including SEQ ID NOs: 39 and 40, apair of primers including SEQ ID NOs: 42 and 43, a pair of primersincluding SEQ ID NOs: 45 and 46, a pair of primers including SEQ ID NOs:48 and 49, a pair of primers including SEQ ID NOs: 51 and 49, a pair ofprimers including SEQ ID NO: 54 and SEQ ID NO: 55, a pair of primersincluding SEQ ID NO: 58 and SEQ ID NO: 59, or a pair of primersincluding SEQ ID NO: 62 and SEQ ID NO: 63.

C. Probe and Primer Variants

Although exemplary probe and primer sequences are provided in SEQ IDNOs: 12-51, 54-56, 58-60, and 62-64, the primer and/or probe sequencescan be varied slightly by moving the probe or primer a few nucleotidesupstream or downstream from the nucleotide positions that they hybridizeto on the target nucleic molecule acid, provided that the probe and/orprimer is still specific for the target nucleic acid sequence, forexample specific for one of SEQ ID NOs: 1-11, 52-53, 57, or 60. Forexample, variations of the probes and primers disclosed as SEQ ID NOs:12-51, 54-56, 58-60, and 62-64 can be made by “sliding” the probesand/or primers a few nucleotides 5′ or 3′ from their positions, and suchvariation will still be specific for the respective target nucleic acidsequence.

Also provided by the present disclosure are probes and primers thatinclude variations to the nucleotide sequences shown in any of SEQ IDNOs: 12-51, 54-56, 58-60, and 62-64, as long as such variations permitdetection of the target nucleic acid molecule. For example, a probe orprimer can have at least 90% sequence identity such as at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleic acidincluding of the sequence shown in any of SEQ ID NOs: 12-51, 54-56,58-60, and 62-64. In such examples, the number of nucleotides does notchange, but the nucleic acid sequence shown in any of SEQ ID NOs: 12-51,54-56, 58-60, and 62-64 can vary at a few nucleotides, such as changesat 1, 2, 3, or 4 nucleotides.

The present application also provides probes and primers that areslightly longer or shorter than the nucleotide sequences shown in any ofSEQ ID NOs: 12-51, 54-56, 58-60, and 62-64, as long as such deletions oradditions permit detection of the desired target nucleic acid molecule,such as one of SEQ ID NOs: 1-11, 52-53, 57, or 60. For example, a probeor primer can include a few nucleotide deletions or additions at the 5′-or 3′-end of the probe or primers shown in any of SEQ ID NOs: 12-51,54-56, 58-60, and 62-64, such as addition or deletion of 1, 2, 3, or 4nucleotides from the 5′- or 3′-end, or combinations thereof (such as adeletion from one end and an addition to the other end). In suchexamples, the number of nucleotides changes.

Also provided are probes and primers that are degenerate at one or morepositions (such as 1, 2, 3, 4, 5, or more positions), for example, aprobe or primer that includes a mixture of nucleotides (such as 2, 3, or4 nucleotides) at a specified position in the probe or primer. In someexamples, the probes and primers disclosed herein include one or moresynthetic bases or alternative bases (such as inosine). In otherexamples, the probes and primers disclosed herein include one or moremodified nucleotides or nucleic acid analogues, such as one or morelocked nucleic acids (see, e.g., U.S. Pat. No. 6,794,499) or one or moresuperbases (Nanogen, Inc., Bothell, Wash.). In other examples, theprobes and primers disclosed herein include a minor groove binderconjugated to the 5′ or 3′ end of the oligonucleotide (see, e.g., U.S.Pat. No. 6,486,308).

V. Kits

The nucleic acid primers and probes disclosed herein can be supplied inthe form of a kit for use in the detection of one or more ofAcinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, or Neisseria meningitidis in a sample.In such a kit, an appropriate amount of one or more of the nucleic acidprobes and/or primers (such as one or more of SEQ ID NOs: 12-51, 54-56,58-60, and 62-64) are provided in one or more containers or in one ormore individual wells of a multiwall plate or card. A nucleic acid probeand/or primer may be provided suspended in an aqueous solution or as afreeze-dried or lyophilized powder, for instance. The container(s) inwhich the nucleic acid(s) are supplied can be any conventional containerthat is capable of holding the supplied form, for instance, microfugetubes, ampoules, or bottles. The kits can include either labeled orunlabeled nucleic acid probes (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 probes) for use in detection of Acinetobacterbaumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasmagondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis,Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonellaspp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acids.One or more control probes and/or primers for use in the PCR reactionsalso may be supplied in the kit. In some examples, the probes aredetectably labeled.

In some examples, one or more sets of primers (such as the primersdescribed above), such as pairs of primers (for example, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pairs of primers), may be providedin pre-measured single use amounts in individual, typically disposable,tubes, wells, or equivalent containers. With such an arrangement, thesample to be tested for the presence of the target nucleic acids can beadded to the individual tube(s) or well(s) and amplification carried outdirectly.

The amount of nucleic acid primer supplied in the kit can be anyappropriate amount, and may depend on the target market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each nucleic acid primer provided wouldlikely be an amount sufficient to prime several PCR amplificationreactions. General guidelines for determining appropriate amounts may befound in Innis et al., Sambrook et al., and Ausubel et al. A kit mayinclude more than two primers in order to facilitate the PCRamplification of a larger number of target nucleic acid molecules, suchas Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellapneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichiacoli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydiatrachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp.,Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidisnucleic acids, or any combination of two or more thereof.

In some embodiments, kits also may include the reagents necessary tocarry out PCR amplification reactions, including DNA sample preparationreagents, appropriate buffers (such as polymerase buffer), salts (forexample, magnesium chloride), deoxyribonucleotides (dNTPs), andpolymerases.

In particular embodiments, the kits include prepackaged probes, such asprobes suspended in suitable medium in individual containers (forexample, individually sealed tubes or wells). In some examples, theprobes include those provided herein. In other particular embodiments,the kit includes equipment, reagents, and instructions for extractingand/or purifying nucleotides from a sample.

The present disclosure is illustrated by the following non-limitingExamples.

Example 1 Primers and Probes

Primers and probes were designed for detection of Acinetobacterbaumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasmagondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcusaureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasmaurealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., andStreptococcus agalactiae. In most cases, oligonucleotides were designedusing Primer Express 3.0 software (Applied Biosystems, Foster City,Calif.) with slight modifications to optimize melting temperatures (Tm)and minimize intra- and inter-molecular interactions. Specificity ofeach set of oligonucleotides for the intended genus and/or species wasassessed by sequence comparison using Basic Local Alignment Search Tool(BLAST) within the National Center for Biotechnology Information (NCBI)database (available on the world wide web atblast.ncbi.nlm.nih.gov/Blast.cgi). Primers and hydrolysis probes usedfor analytical validation were manufactured by the Biotechnology CoreFacility at the Centers for Disease Control and Prevention (Atlanta,Ga., USA).

The target genes and primer and probe sequences are shown in Table 1.Some probes (such as the P. jirovecii, U. parvum, and Ureaplasma spp.probes) include a BHQ1 internal quencher linked to a “T” nucleotide.Probes including an internal quencher include C6 CpG at the 3′ end toprevent extension. In some examples, each probe includes 5′ FAM labeland a 3′ BHQ1 label, except where indicated otherwise.

TABLE 1  Primers and probes for detection of pathogens SEQ Final IDConc. Organism Target Oligo Sequence NO: (nM) Acinetobacter oxa-51 ABF1TATTTTTATTTCAGCCTG 12 1000 baumannii CTCACCTT ABR3 AAATACTTCTGTGGTGGT 131000 TGCCTTA ABP1 TGACTGCTAATCCAAATC 14 200 ACAGCGCTTCA Pseudomonas gyrBPAF2 GTCTCGGTGGTGAACG 15 500 aeruginosa PAR TGGATGTTGCTGAAGGTC 16 500 TCPAP2 TCCGTCGCCACAACAAGG 17 100 TCTGGGAA Klebsiella nifA KPF1TGCTGCATAAAGGCATCG 18 1000 pneumoniae TT KPR1 CCACCGAGGCCAGCAA 19 1000KPP1 ACGCTGAGCACCTCCTGC 20 200 AACGT diguanylate For TGCAGATAATTCACGCCC58 1000 cyclase AG Rev ACCCGCTGGACGCCAT 59 1000 Probe CCACCACGCTCATCTGTT60 200 TCGCC Toxoplasma ssrRNA TGF2 GGTGGTCCTCAGGTGAT 21 1000 gondiiTGR2 CCACGGTAGTCCAATACA 22 1000 GTA TGP2 ATCGCGTTGACTTCGGTC 23 200TGCGAC Moraxella purH MCF2 GGTGAGTTGCCACAGC 24 1000 catarrhalis MCR2AGTAGACCGCCATTGACT 25 1000 C MCP1 CACAGCGGGCAGCTCAAT 26 200 TTGACCTAEscherichia uidA ECSF1A GAGCATCAGGGTGGCTAT 27 500 coli/ ACG ShigellaECSR1A ATAGTCTGCCAGTTCAGT 28 500 TC ECSP1 TACGGCGTGACATCGGCT 29 100TCAAATG Staphylococcus gsf SAF2 CGGGTTAGGTGAATTGAT 30 1000 aureusTGTTTTAT SAR2 CGCATTTGAGCTGAAGTT 31 1000 G SAP2 TTCCATATGACCACCACG 32200 AGTCTTAGCACC Pneumocystis dhps PJF1 TAATGGTTTGCCTTGGTT 33 1000jirovecii GCTT PJR2A CACAGCCTCCTAAAACAG 34 1000 AT PJP2AACAGGGTGTCT“T”ACAG 35 200 GTGATGTTATGCCAAAAG Chlamydia tmRNA CTF1GGTGTAAAGGTTTCGACT 36 1000 trachomatis TAGAA CTR3 CGAACACCGGGTCACC 371000 CTP1 ATGCGGAGGGCGTTGGCT 38 200 GG Ureaplasma mba UUF1ATTTCATATTTAGTTTAT 39 1000 urealyticum TAGGAGATCGTTAT UUR1AGATTTAACATTTGAGCT 40 1000 AGAACAT UUP2 CACAGCAACTACCCCTGC 41 200TCCCACTAA Ureaplasma ure PARVUMF3 ACAGATAATGTTGATATG 42 1000 parvumATTGTGGGTAT PARVUMR3 CTAATGCAACAGGAACTA 43 1000 TTTCTG PARVUMP3iTCAGTGAACG“T”GAGTA 44 200 TCTAAACCACCAGC Ureaplasma ure UPANF1GGTTTAGATACTCACGTT 45 500 spp. CACTGA UPANR1 GCTTTTGTACCATCATTC 46 500ATACCTGT UPANP1i CCACCAGCAA“T”AACAG 47 100 TTGTAATACCACCATC BartonellassrA ssrA-F GCTATGGTAATAAATGGA 48 500 spp. CAATGAAATAA ssrA-RGCTTCTGTTGCCAGGTG 49 500 ssrA-P ACCCCGCTTAAACCTGCG 50 100 ACG ssrA-F2CTAAATGAGTAGTTGCAA 51 ATGACAAC Streptococcus cfb For GGGAACAGATTATGAAAA54 1000 agalactiae ACCG Rev AAGGCTTCTACACGACTA 55 1000 CCAA ProbeAGACTTCATTGCGTGCCA 56 200 ACCCTGAGAC Neisseria sodC ForCTGTGAGCCAAAAGAAAA 62 1000 meningitidis AGAAG Rev GATTTGTTGCTGTGCCAT 631000 CAT Probe CGCAGGCGGTCACTGGGA 64 200 TC Salmonella ttrRSBCA ForCTCACCAGGAGATTACAA 65 500 spp. CATGG Rev AGCTCAGACCAAAAGTGA 66 500 CCATCProbe CACCGACGGCGAGACCGA 67 100 CTTT “T” = BHQ1 modification

Example 2 Detection and Differentiation of Bartonella Species andGenotypes

Bacterial Strains and DNA Extraction:

All bacterial strains were obtained from collections at the Centers forDisease Control and Prevention in Fort Collins, Colo. and Atlanta, Ga.Nucleic acid was extracted from 33 Bartonella strains, including 25defined species or subspecies using the QIAAMP DNA mini kit (Qiagen,Valencia, Calif.). Bartonella strains included in this study were: B.alsatica (IBS 382), B. bacilliformis (KC584), B. birtlesii (IBS 325), B.bovis (91-4), B. capreoli (WY-Elk), B. chomelii (A828), B. clarridgeiae(Houston-2), B. doshiae (R18), B. elizabethae (F9251), B. henselae(Houston-1), B. grahamii (V2), B. japonica (Fuji 18-1T), B. koehlerae(C-29), B. melophagi (K-2C), B. phoceensis (16120), B. quintana(Fuller), B. rochalimae (BMGH), B. schoenbuchensis (R1), B. silvatica(Fuji 23-1T), B. tamiae (Th307, Th239, and Th339), B. taylorii (M16), B.tribocorum (IBS 506), B. vinsonii subsp. arupensis (OK 94-513), B.vinsonii subsp. vinsonii (Baker), B. washoensis (Sb944nv), andBartonella isolates (Sh6397ga, Sh6396ga, Sh6537ga, Sh8784ga, Sh8200ga,and Sh8776ga). Using the MAGNA PURE® Compact instrument with TotalNucleic Acid Isolation Kit I (Roche Applied Science, Indianapolis,Ind.), nucleic acid was extracted from 61 microorganisms that areclosely related genetically to Bartonella or may occupy a similarecological niche, including Afipia broomii, Afipia clevelandensis,Afipia felis, Agrobacterium radiobacter, Agrobacterium tumefaciens,Babesia microti, Bordetella pertussis, Bordetella parapertussis,Bradyrhizobium, Brucella abortus, Brucella canis, Brucella melitensis,Brucella neotomae, Brucella ovis, Brucella suis, Campylobacter coli,Campylobacter fetus, Campylobacter jejuni, Citrobacter freundii,Enterobacter aerogenes, Enterobacter cloacae, Erwinia, Escherichiaalbertii (2 strains), Escherichia blattae, Escherichia coli (4 strains),Escherichia fergusonii, Escherichia hermanii, Escherichia vulneris,Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae,Kluyvera intermedia, Legionella pneumophila, Methylobacteriumorganophilum, Ochrobactrum anthropi (3 strains), Ochrobactrumintermedium, Oligella urethralis (4 strains), Psychrobacterphenylpyruvicus (2 strains), Raoultella planticola, Salmonella bongori,Salmonella enterica (serovar Enteriditis, serovar Typhi, serovarTyphimurium), Shigella boydii, Shigella dysenteriae, Shigella flexneri,Shigella sonnei, Toxoplasma gondii, and Vibrio cholerae. Human genomicDNA was also tested for cross-reactivity. All nucleic acid extracts werenormalized to 1 ng/μL in Tris-EDTA buffer.

Real-Time PCR:

Sequences of the ssrA (tmRNA) gene of five representative Bartonellaspecies were obtained from the tmRNA Website (indiana.edu/˜tmrna/) andGenBank (accession numbers: NC_(—)005955.1, NC_(—)005956.1,NC_(—)010161.1, NC_(—)012846.1, NC_(—)008783.1). Sequences were alignedusing the Clustal W method (ebi.ac.uk/Tools/msa/clustalw2/). Primers andprobes were designed using Primer Express 3.0 software (AppliedBiosystems, Foster City, Calif.) with some modification foramplification of a 301 bp region of ssrA. The reaction mix (25 μL)contained the following components: 12.5 μL 2× PerfeCta® MultiPlex qPCRSuperMix (Quanta Biosciences, Gaithersburg, Md.), forward and reverseprimers (ssrA-F: 5′-GCTATGGTAATAAATGGACAATGAAATAA-3′ (SEQ ID NO: 48),ssrA-R: 5′-GCTTCTGTTGCCAGGTG-3′ (SEQ ID NO: 49)) at a finalconcentration of 500 nM, FAM-labeled probe(5′FAM-ACCCCGCTTAAACCTGCGACG-3′BHQ1 (SEQ ID NO: 50)) at a finalconcentration of 100 nM, and 5 μL of extracted nucleic acid. Real-timePCR was performed on the Applied Biosystems 7500 real-time PCRinstrument with the following thermocycling parameters: 1 cycle of 95°C. for 2 min followed by 45 cycles of 95° C. for 15 sec and 60° C. for60 sec with data collection in the FAM channel. Primers and probe weretested using nuclease-free water (n=95) to ensure no signal in theabsence of nucleic acid template. The limit of detection wasindependently determined and verified for four species (B. quintana, B.henselae, B. bovis, and B. elizabethae) by testing 10 replicates each of10-fold serial dilutions of genomic DNA ranging from 1 ng/μL to 0.1fg/μL. The limit of detection was identified as the lowest dilution atwhich amplification was observed in at least 50% of replicates.Specificity was assessed by performing the assay using 15 ng of nucleicacid from 61 different microorganisms representing 24 genera and 48species.

Sequencing:

Amplicons for sequencing were generated by conventional PCR with forwardand reverse primers at 400 nM each using the Bio-Rad Dyad® thermalcycler (Bio-Rad, Hercules, Calif.) with the following thermocyclingconditions: 95° for 2 min, 30 cycles of 95° for 15 sec, 60° for 60 sec,and 72° for 30 sec followed by 72° for 3 min. Amplicons were visualizedby electrophoresis in a 1% agarose gel followed by staining with 0.05%methylene blue solution and purification using the Geneclean® Turbo kit(MP Biomedicals, Solon, Ohio). Sequencing reactions were performed inboth directions using BigDye® Terminator 3.1 cycle sequencing kit(Applied Biosystems) according to the manufacturer's instructions withthe same primers for the real-time PCR assay at a final concentration of165 nM. Sequencing was performed on the Applied Biosystems 3130xLgenetic analyzer.

Phylogenetic Analysis:

A 253 bp region of each amplified sequence (excluding forward andreverse primers) was used for alignment and phylogenetic comparison ofBartonella species and genotypes using Lasergene® version 8 softwaresuite (DNASTAR, Madison, Wis.). All ssrA sequences were aligned usingthe Clustal V method. Phylogenetic trees were constructed using theneighbor joining method and bootstrapping analysis with 1,000replicates.

Testing of Animal Blood:

Blood specimens collected from elk (Cervus elaphus) in Wyoming (n=56)and cattle (Bos primigenius) in the country of Georgia (n=89) between2008-2009 were tested for Bartonella by bacterial culture usingpreviously described methods (Bai et al., Vet. Microbiol. 148:329-332,2011). The culture results from this cohort of elk have been reportedpreviously (Bai et al., 2011). All specimens were extracted using theDNEASY® Blood and Tissue kit (Qiagen) or MAGNA PURE® Compact with TotalNucleic Acid Isolation Kit I (Roche Applied Science). Five or 10 μL ofnucleic acid extract was used in each real-time PCR reaction.

Nucleotide Sequence Accession Numbers:

Thirty-four unique ssrA sequences obtained from Bartonella strains andisolates were submitted to GenBank and assigned the following accessionnumbers: JNO29776 (B. alsatica IBS 382), JNO29794 (B. bacilliformisKC584), JNO29775 (B. birtlesii IBS325), JNO29767 (B. bovis 91-4),JNO29798 (B. capreoli WY-Elk), JNO29773 (B. chomelii A828), JNO29768 (B.doshiae R18), JNO29774 (B. elizabethae F9251), JNO29785 (B. henselaeHouston-1), JNO29795 (B. grahamii V2), JNO29784 (B. japonica Fuji18-1T), JNO29769 (B. koehlerae C-29), JNO29771 (B. melophagi K-2C),JNO29770 (B. phoceensis 16120), JNO29766 (B. quintana Fuller), JNO29797(B. rochalimae BMGH), JNO29772 (B. schoenbuchensis RD, JNO29782 (B.silvatica Fuji 23-1T), JNO29778 (B. tamiae Th307), JNO29779 (B. tamiaeTh239), JNO29780 (B. tamiae Th339), JNO29781 (B. taylorii M16), JNO29796(B. tribocorum IBS 506), JNO29783 (B. vinsonii subsp. arupensis OK94-513), JNO29777 (B. vinsonii subsp. vinsonii Baker), JNO29786 (B.washoensis Sb944nv), JNO29787 (Bartonella sp. Sh6397ga), JNO29791(Bartonella sp. Sh8200ga), JNO29793 (Bartonella sp. Sh8776ga), JNO29788(Bartonella sp. Sh6396ga), JNO29790 (Bartonella sp. Sh8784ga), JNO29792(Bartonella sp. Sh9282ga), JNO29789 (Bartonella sp. Sh6537ga), JN982716(B. clarridgeiae Houston-2). The ssrA sequence amplified from elk bloodwas assigned accession number JN982717, and the sequence identified incattle blood was identical to B. bovis (JNO29767).

Real-Time PCR for Detection of Bartonella ssrA:

Amplification of the target sequence occurred with all Bartonellaspecies (n=24) and unclassified Bartonella strains (n=7) tested (datanot shown). Amplification curves demonstrated sigmoidal shape and hadcrossing threshold (Ct) values between 15 and 21 with 5 ng of DNA perreaction. No amplification was observed in no-template control (NTC)reactions (n=95) or with DNA from other microorganisms listed above(n=61) or human genomic DNA. The limit of detection was independentlydetermined for four species (B. quintana, B. henselae, B. bovis, and B.elizabethae) and found to be ≦5 fg of nucleic acid per reaction.

Bartonella Phylogeny Based on ssrA Genotypes:

Phylogenetic analysis of ssrA sequences from each Bartonella strain orisolate showed that this region was sufficient to discriminate allBartonella species and that separation of clades based on ssrA sequenceswas consistent with phylogeny based on gltA, which is considered areliable tool for distinguishing closely related Bartonella genotypes(LaScola et al., Trends Microbiol. 11:318-321, 2003). First, the ssrAsequences from ruminant-associated Bartonella, including B. chomelii,163 B. capreoli, B. bovis, B. melophagi, and B. schoenbuchensis formedan independent clade; sequence identity between these species was ≧94%.Further, both subspecies of B. vinsonii (vinsonii and arupensis)included in this study formed a separate grouping in the tree with 98%identity, as did three strains of the recently identified pathogenicBartonella species B. tamiae (≧97.2% identity) (Kosoy et al., J. Clin.Microbiol. 46:772-775, 2008). Among all ssrA sequences, the lowestpercent identity (75.3 to 84.1%) was observed for strains of B. tamiaerelative to other Bartonella species, thus supporting the separation ofB. tamiae as a novel species (Kosoy et al., 2008). The division of twoadditional clades which are similarly separated by gltA comparison, oneconsisting of B. elizabethae and B. tribocorum and the other includingB. henselae and B. koehlerae, were also supported by the phylogeneticanalysis of ssrA. Overall, the separation of major Bartonella cladesbased on ssrA sequences was consistent with phylogeny based on gltA(Kosoy et al., Am. J. Trop. Med. Hyg. 82:1140-1145, 2010; Maillard etal., Int. Syst. Evol. Microbiol. 54:215-220, 2004).

Detection and Identification of Bartonella in Animal Blood:

This assay was used to screen elk and cattle blood specimens for thepresence of Bartonella and compared to bacterial culture results.Bartonella DNA was detected in 16 of 55 (29.1%) and 42 of 89 (47.2%)specimens from elk and cattle, respectively. The appropriate ampliconsize was confirmed for positive samples. Using traditional culturingmethods, Bartonella was recovered from only 4 of 55 (7.3%) elk and 34 of89 (38.2%) cattle specimens. Since comparison of ssrA genotypes fromBartonella reference strains showed that this sequence providessufficient information to discriminate Bartonella genotypes, weperformed sequencing analysis of a subset of ssrA sequences amplifiedfrom elk (n=3) and cattle (n=5) specimens in order to identify theBartonella species present. Analysis of ssrA sequences from elk bloodrevealed one genotype which clustered most closely with B. capreoli, aBartonella species found in wild and domestic ruminants (Bai et al.,Vet. Microbiol. 148:329-332, 2011). These results were consistent withprevious identification of B. capreoli isolated from these samples usingsequencing analysis of gltA (Bai et al., 2011). Similarly, a single ssrAgenotype present in cattle blood was found to be identical to B. bovis(99.7% similarity). This result corroborated previous identification ofB. bovis from these cattle specimens by analysis of gltA.

Samples from four patients in Thailand presenting with headache,myalgia, dizziness, fatigues, and rat exposure and animal ownership wereanalyzed for Bartonella infection. Blood clots from each patient wereinoculated into Bartonella alpha-Proteobacteria growth medium andincubated aerobically at 35° C. with 5% CO₂ for seven days. DNA wasextracted from this pre-enrichment using the QIAAMP® DNA mini kit(Qiagen, Chasworth, Calif.) according to manufacturer's instruction, andanalyzed using real-time PCR targeting ssrA, as described above. PCRassays were performed using a CFX96™ Real-Time System (Bio-Rad,Hercules, Calif.). Amplicons were recovered from PCR reactions bygel-purification and sequenced in both directions using an AppliedBiosystems Model 3130 Genetic Analyzer (Applied Biosystems, Foster City,Calif.).

Sequences obtained from the patients were very similar to the typestrain of B. vinsonii subsp. arupensis. The ssrA sequences revealed twosimilar variants. One variant was identical to the type strain of B.vinsonii. subsp. arupensis (JNO29783) and was identified in three of thepatients (45-00250, 45-01217, and 45-01239). The other variant(JN394654), from patient 45-00025, was 2.8% divergent from the typestrain of B. vinsonii. subsp. arupensis.

Example 3 Multiple Pathogen Detection in Population-Based Study ofNeonatal Infection Materials and Methods

Real-Time PCR Assay Design and Analytical Validation:

A panel of neonatologists with expertise in neonatal infection and SouthAsia was convened, and the Delphi method (Dalkey and Helmer, ManagementSci. 9:458-467, 1963) was used to identify organisms of the highestpriority for testing in nasopharyngeal (NP) and oropharyngeal (OP) swabsand blood specimens from neonates. Primers and hydrolysis probes weredesigned as described in Example 1.

All newly developed real-time PCR assays were evaluated using individualRT-qPCR reactions prior to use on the TAQMAN array card (TAC) format.Each assay was tested using nuclease-free water as template (n≧95) toensure no fluorescence amplification signal was observed in the absenceof nucleic acid. The limit of detection was independently determined foreach assay by testing at least 3 replicates each of a 10-fold dilutionseries of specific total nucleic acid ranging from 0.1 fg/μL to 1 ng/μL.Inclusivity was assessed by testing representative isolates, includingvarious subspecies, serotypes, or clonal groups, as appropriate (Table2). Specificity of each assay was assessed by testing 15 ng of nucleicacid from at least 200 different bacteria, viruses, and protozoarepresenting 36 genera and 143 species. In addition to the most closelyrelated species to each target pathogen, this panel also includedcommensals of the respiratory tract and human nucleic acid.

Oligonucleotide Preparation for TAC Manufacturing:

Oligonucleotides for TAC production were manufactured by Integrated DNATechnologies (Coralville, Iowa) or Biosearch Technologies (Novato,Calif.), diluted and combined to 20× reaction concentration, andprovided to Life Technologies (Foster City, Calif.) for custommanufacturing of study-specific TACs. The 20× concentration correspondsto a 1× final reaction concentration in the 1 μL reaction within eachTAC well on the finished card. Total nucleic acid was extracted from aseries of 10-fold dilutions of each organism and tested to determine thepotential impact of oligonucleotide concentration on assay sensitivity.

Clinical Specimens:

Clinical specimens, including whole blood and combined NP/OP swabs, wereobtained from enrolled neonates at three ANISA study sites: Sylhet,Bangladesh; Karachi, Pakistan; and Matiari, Pakistan. NP/OP swabs werecollected and placed together in 1 mL Universal Transport Media (UTM,Copan Diagnostics, Murrieta, Calif.) and stored at −70° C. prior toextraction. Blood specimens were collected in standard EDTA collectiontubes and stored at 4° C. for short-term storage (≦72 h post-collection)or −70° C. for longer storage prior to nucleic acid extraction andtesting by TAC. Additional respiratory clinical specimens (NP/OP swabs)from the historical collection at the Centers for Disease Control andPrevention (Atlanta, Ga.) were also used for some experiments.

Specimen Processing and Nucleic Acid Extraction:

Total nucleic acid (TNA) was extracted from clinical specimens using theMAGNA PURE® Compact instrument (Roche Applied Sciences, Indianapolis,Ind.) with Nucleic Acid Isolation Kit I and Total NA Plasma protocol.For NP/OP swab specimens, 400 μL of UTM was extracted and eluted in 100μL. For extraction of whole blood, 300 μL of blood in EDTA was mixedwith 100 μL of a freshly-prepared solution of lytic enzymes consistingof 1.5 mg/mL lysostaphin, 2500 U/mL mutanolysin, and 200 mg/mL lysozyme(Sigma-Aldrich, St. Louis, Mo.) in Tris-EDTA (TE) buffer and incubatedat 37° C. for 30-60 min. prior to extraction on the MAGNA PURE® Compact,with elution in 100 μL. To assess the potential impact of a pre-lysisstep on recovery of TNA from a variety of pathogens, healthy donor bloodwas spiked with 10-fold serial dilutions of quantified culture stock ofgram-positive bacteria (S. aureus), gram-negative bacterium (K.pneumoniae), or an RNA virus (enterovirus) and tested using individualRT-qPCR reactions. Spiked blood specimens were extracted directly orincubated with TE buffer or TE buffer containing lytic enzymes at 37° C.for 30 min. prior to extraction. Ct values for spiked blood experimentswere compared using Student's two-tailed t test.

Individual Real-Time PCR Assay Performance:

All individual real-time PCR assays were performed on the AppliedBiosystems 7500 Real-Time PCR system (Life Technologies, Foster City,Calif.) with the following cycling conditions: 45° C. for 10 minutes,94° C. for 10 minutes, 45 cycles of 94° C. for 30 seconds and 60° C. for60 seconds, with data acquisition in the FAM channel during the 60° C.step. Each reaction consisted of 1× AGPATH-ID™ One-step RT-PCR bufferand 1× AGPATH-ID™ One-step RT-PCR enzyme mix (Applied Biosystems, FosterCity, Calif.) or 1× QSCRIPT™ XLT One-step RT-qPCR TOUGHMIX®, low ROX(Quanta Biosciences, Gaithersburg, Md., USA), forward and reverseprimers and FAM-labeled hydrolysis probe at the concentrations listed inTable 1, and nuclease-free water to final volume of 20 μL. Five μL ofTNA was used in each reaction.

TAC Assay Performance:

Mastermix for each TAC consisted of the following: 1× AGPATH-ID™One-step RT-PCR buffer and enzyme or 1× QSCRIPT™ XLT One-step RT-qPCRTOUGHMIX®. Reactions tested using AGPATH-ID™ enzyme system consisted of50 μL 2× buffer, 4 μL 25× enzyme mix, and 46 μL of TNA. Reactions testedwith the QSCRIPT™ enzyme system consisted of 50 μL 2× mastermix and 50μL of TNA. Each card was centrifuged at 336×g for 1 min. twice, todistribute the fluid in the reaction wells, and sealed to sequesterindividual reactions. All TACs were run on the Applied Biosystems VIIA™7 Real-Time PCR system (Life Technologies, Foster City, Calif.) usingthe same cycling conditions as used for individual RT-qPCR reactions. Ano template control (NTC) and a positive control consisting of combinedRNA transcripts generated as previously described (Kodani and Winchell,J. Clin. Microbiol. 50:1057-1060, 2012) were included on each card.

Results

Analytical Validation of New Real-Time PCR Assays:

For each new assay, no amplification was observed in no-template control(NTC) reactions (n≧95) or in reactions containing nucleic acid fromother organisms (n≧200, data not shown). Each assay was also tested forinclusivity within the genus or species using representative isolates ofeach subspecies or serotype as appropriate (Table 2). The number ofisolates used for inclusivity testing varied based on availability. Thelimit of detection was independently determined for each assay (Table2).

TABLE 2 Analytical validation of newly developed real-time PCR assaysfor ANISA study No. of Limit of isolates Assay Detection tested Notes T.gondii  <1 fg 1 S. aureus 100 fg-10 fg/μL 11 Tested representativeisolates from various MRSA clonal groups (USA 100, 200, 300, 400, 500,800, 1000, Brazilian, EMRSA 15, and ST80) K. pneumoniae 100 fg/μL 2 E.coli and 10 fg-1 fg/μL 94 Tested 21 Shigella isolates (4 differentShigella spp. spp.) P. aeruginosa 100 fg-10 fg/μL 7 Ureaplasma spp.  1pg/μL 14 Tested representative strain of all serotypes of U. urealyticumand U. parvum (1-14) C. trachomatis 15 copies/μL 4 Tested representativeisolates of serovars D, E, H, and F A. baumannii 10 fg-1 fg/μL 1 S.agalactiae 300 fg/μL 30 Tested representative isolates from all (GBS)serotypes (1A, 1B, 2-7) and non- typeable isolates (n = 2)

The target for real-time PCR detection of E. coli also reacts with theShigellae. This E. coli/Shigella assay successfully detected all E. colitypes tested, including representative isolates of each virotype (EHEC,EPEC, ETEC, EAEC, and EIEC), but did not amplify the closely relatedspecies E. albertii, E. hermannii, or E. fergusonii. This assay alsodetected all four Shigella species (S. flexneri, S. sonnei, S.dysenteriae, and S. boydii), with the exception of S. dysenteriaeserotype I. During development and validation of this assay, sporadicamplification signal in NTC reactions was occasionally observed. Thiswas determined to occur due to residual E. coli DNA present in theenzyme preparation from the manufacturers, which varied betweenproduction lots. Residual E. coli DNA in extraction reagents may alsocontribute to this phenomenon during testing of clinical specimens.Crossing threshold (Ct) values for this sporadic amplification weregenerally found to be >30. For this reason, a Ct cutoff value of 30 wasimplemented.

Extraction of TNA from Blood and Saline:

Direct comparison of Ct values of TNA extracted from blood spiked withgram-positive bacteria (S. aureus) revealed that the average Ct valuewas approximately 5.5 cycles lower for TNA extracted after incubationwith pre-lysis enzymes compared to identical preparations without thispre-treatment step (FIG. 1A). Incubation of spiked blood specimens withTE buffer alone did not result in lower Ct values, indicating that theobserved improvement in Ct values was a result of the pre-lysis enzymetreatment instead of simply dilution and heating. Pre-treatment withlytic enzymes had no significant impact on Ct values of TNA from bloodspiked with the gram-negative bacterium K. pneumoniae (FIG. 1B).

In addition, pre-lysis did not significantly impact target detection insaline spiked with the same serial dilutions. However, comparison of theCt values for TNA extracted from blood and saline spiked with the sameconcentration of organisms revealed that detection in blood issignificantly impaired relative to saline. The difference in Ct valuefor detection of the same number of bacteria in blood compared to salineranged from 3.8 cycles at higher concentrations to 11.8 cycles at lowerconcentrations (mean difference in Ct value=6), including a completelack of detection of the lowest concentration in blood (FIG. 1C).

TAC Preparation: Oligonucleotide Concentration and Assay Replicates:

The impact of oligonucleotide concentration on pathogen detection wasbriefly examined using the optimization TAC configuration. Nosignificant difference in target amplification was observed at the limitof detection for any of the four targets examined. Additionally, testingof clinical specimens previously known to be positive for each of thesepathogens did not reveal any oligonucleotide concentration-dependentdifference in target detection.

Testing of serial dilutions of nucleic acid from M. pneumoniae, S.pneumoniae, S. agalactiae, and PIV2 revealed excellent concordancebetween replicates at higher TNA concentrations. In contrast, the numberof replicate reactions in which amplification was observed decreased asthe concentration approached the limit of detection for each assay.Testing five replicates allowed detection of less concentrated nucleicacid compared to two replicates. These results indicate that testing ahigher number of replicates improves pathogen detection rates when theconcentration of organisms is near the limit of detection.

Positive results in clinical specimens were confirmed by repeat testingin individual RT-qPCR reactions followed by confirmation of theappropriate size amplicon. The proportion of NP/OP and blood specimensidentified as positive in more than half of assay replicates varied bytarget (FIG. 2). Overall, this proportion was significantly higher inNP/OP specimens (FIG. 2A) compared to whole blood (FIG. 2B). In otherwords, while the majority of NP/OP specimens that were positive wereidentified as positive in more than half of assay replicates,amplification of pathogen-specific targets in whole blood extractsoccurred in a much smaller proportion of total replicates tested.Furthermore, average Ct values were higher in positive reactionscontaining TNA extracted from blood compared to NP/OP swabs. Togetherthese data suggest that a higher number of assay replicates may identifypathogens which otherwise would be missed, particularly in whole bloodspecimens.

Enzyme System Performance with TAC:

Newer generations of enzyme mixes may result in improved detection ofpathogen targets, particularly in the presence of molecules known tohave inhibitory effects on real-time PCR, such as some blood components.The performance of two enzyme formulations, Ambion AGPATH-ID™ One-stepRT-PCR kit (Applied Biosystems) and Quanta QSCRIPT™ XLT One-step RT-qPCRTOUGHMIX®, low ROX (Quanta Biosciences), at detecting targets in NP/OPspecimens (n=18) and blood specimens (n=12) (FIG. 3) were compared. Themedian difference in Ct value for positive results in NP/OP specimenstested with TOUGHMIX® compared to AGPATH-ID™ enzyme varied by target(range 0-4), but overall improved Ct values were observed in reactionsusing TOUGHMIX® enzyme (FIG. 3A). An even more dramatic improvement inCt values was observed for blood specimens tested with TOUGHMIX®compared to AGPATH-ID™; the median difference in Ct value ranged from0.46 to 10.5 for various targets (FIG. 3B). In addition, additionalpositive results (n=16) for pathogen-specific targets were detectedusing the TOUGHMIX® enzyme mix that were not detected when testing thesame specimen extract using AGPATH-ID™. This phenomenon was not limitedto a single pathogen target, but rather occurred with 12 unique assays.While a few instances (n=5) were also observed where the reactions usingAGPATH-ID™ yielded a positive result while the TOUGHMIX® reaction wasnegative, this occurred only when the Ct value with AGPATH-ID™ was >33,at the threshold where reproducibility between replicates is mostcommonly discordant. Overall, improved pathogen detection was observedusing the TOUGHMIX® enzyme system, particularly in primary bloodspecimens.

Example 4 Diagnostic Multiplex PCR Assay

This example describes exemplary methods that can be used to detect oneor more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellapneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichiacoli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydiatrachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp.,Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidisnucleic acids in a sample from a subject, thereby diagnosing the subjectwith infection with the detected organism(s). The methods can also beused to detect presence of one or more of Acinetobacter baumannii,Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii,Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus,Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum,Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcusagalactiae, and/or Neisseria meningitidis in an environmental sample.One of ordinary skill in the art will appreciate that methods thatdeviate from these specific methods can also be used to successfullydetect Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellapneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichiacoli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydiatrachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp.,Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidisnucleic acids in a sample.

Clinical samples are obtained from a subject (such as a subjectsuspected of having a CAP infection), such as a nasopharyngeal,oropharyngeal, or bronchial swab, bronchoalveolar lavage, or sputum, oran environmental sample is obtained, for example by swabbing a surfacesuspected of harboring one or more pathogens. DNA is extracted from thesample using routine methods (for example using a commercial kit).

Multiplex real-time PCR is performed in a reaction including a reactionmix (e.g., buffers, MgCl₂, dNTPs, and DNA polymerase), sample DNA (5 μlof nucleic acid extracted from the sample), and probes and primers (suchas those in Table 1, above). The probes and primers are included in thereaction at concentrations of about 25 nM to 1 μM. The assay isperformed using a real-time PCR system (such as the ABI 7500). Exemplarythermocycling conditions are 5 minutes at 95° C., followed by 45 cyclesof 95° C. for 15 seconds and 60° C. for 1 minute. Positive samples arethose with a positive C_(t) value for one or more pathogen probes.

Example 5 Diagnostic Microfluidic Card Assay

This example describes exemplary methods that can be used tosimultaneously detect one or more of Acinetobacter baumannii,Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii,Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus,Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum,Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcusagalactiae, and/or Neisseria meningitidis nucleic acids in a sample froma subject, thereby diagnosing the subject with infection with thedetected organism(s), or presence of the detected organism(s) in anenvironmental sample. One of ordinary skill in the art will appreciatethat methods that deviate from these specific methods can also be usedto successfully detect one or more of Acinetobacter baumannii,Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii,Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus,Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum,Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcusagalactiae, and/or Neisseria meningitidis nucleic acids in a sample.

Clinical samples are obtained from a subject (such as a subjectsuspected of having a pathogenic infection), such as a nasopharyngeal,oropharyngeal, or bronchial swab, bronchoalveolar lavage, or sputum, oran environmental sample is obtained, for example by swabbing a surfacesuspected of harboring one or more pathogens. Nucleic acids (such asDNA, RNA, or total nucleic acid) are extracted from the sample usingroutine methods (for example using a commercial kit).

A microfluidic card (such as a TAQMAN® Array card (also known as aTAQMAN® Low Density Array card); Applied Biosystems, Foster City,Calif.) including primers and probes for one or more of Acinetobacterbaumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasmagondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcusaureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasmaurealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp.,Streptococcus agalactiae, and/or Neisseria meningitidis is utilized.Individual wells of the card include primers and probe for a singlepathogen, which are preloaded and dried onto the designated wells (forexample in duplicate). The card may include at least one well containingAcinetobacter baumannii primers and probe (SEQ ID NOs: 12-14), at leastone well containing Pseudomonas aeruginosa primers and probe (SEQ IDNOs: 15-17), at least one well containing Klebsiella pneumoniae primersand probe (SEQ ID NOs: 18-20 and/or SEQ ID NOs: 58-60), at least onewell containing Toxoplasma gondii primers and probe (SEQ ID NOs: 21-23),at least one well containing Moraxella catarrhalis primers and probe(SEQ ID NOs: 24-26), at least one well containing E. coli/Shigellaprimers and probe (SEQ ID NOs: 27-29), at least one well containingStaphylococcus aureus primers and probe (SEQ ID NOs: 30-32), at leastone well containing Pneumocystis jirovecii primers and probe (SEQ IDNOs: 33-35), at least one well containing Chlamydia trachomatis primersand probe (SEQ ID NOs: 36-38), at least one well containing Ureaplasmaurealyticum primers and probe (SEQ ID NOs: 39-41), at least one wellcontaining Ureaplasma parvum primers and probe (SEQ ID NOs: 42-44), atleast one well containing Ureaplasma spp. primers and probe (SEQ ID NOs:45-47), at least one well containing Bartonella spp. primers and probe(SEQ ID NOs: 48-50 or SEQ ID NOs: 49-51), at least one well containingStreptococcus agalactiae primers and probe (SEQ ID NOs: 54-56), or atleast one well containing Neisseria meningitidis primers and probe (SEQID NOs: 62-64). Each probe includes a 5′ FAM fluorophore. Unlessotherwise noted, each probe also includes a 3′ BHQ1 quencher. The P.jirovecii probe (SEQ ID NO: 35), the U. parvum probe (SEQ ID NO: 44),and the Ureaplasma spp. probe (SEQ ID NO: 47) each also include aninternal BHQ1 quencher. One of ordinary skill in the art can selectdifferent labels and quenchers with only routine testing.

A master mix, including 1× RT-PCR buffer, 1× RT-PCR enzyme and nucleicacids isolated from a sample is applied to the microfluidic cardutilizing the loading ports. The cards are centrifuged, sealed, andplaced in a thermocycler (such as the Applied Biosystems VIIA™ 7real-time PCR platform). Cycling conditions are 45° C. for 10 minutes,94° for 10 minutes, and 45 cycles of 94° C. for 30 seconds and 60° C.for 1 minute (although these conditions can be adjusted by one ofordinary skill in the art to obtain optimal results, for example 55° C.,instead of 60° C., in some examples). Positive samples are those with apositive C_(t) value for one or more probes.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

1. A method for detecting presence of a pathogen selected from the groupconsisting of Pseudomonas aeruginosa, Pneumocystis jirovecii, Bartonellaspp., Acinetobacter baumannii, Klebsiella pneumoniae, Toxoplasma gondii,Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcusaureus, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasmaparvum, Ureaplasma spp., Streptococcus agalactiae, and a combination oftwo or more thereof in a sample, comprising: contacting the sample withone or more nucleic acid probes between 20 and 40 nucleotides in lengthcomprising a nucleic acid sequence set forth as any one of SEQ ID NOs:14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 56, 60, or thereverse complement thereof; and detecting hybridization between the oneor more probes and a nucleic acid in the sample, wherein detection ofhybridization indicates the presence of one or more of said pathogens inthe sample.
 2. The method of claim 1, wherein: the pathogen isPseudomonas aeruginosa and the probe comprises or consists of thenucleic acid sequence set forth as SEQ ID NO: 17; the pathogen isPneumocystis jirovecii and the probe comprises or consists of thenucleic acid sequence set forth as SEQ ID NO: 35; the pathogen isBartonella spp. and the probe comprises or consists of the nucleic acidsequence set forth as SEQ ID NO: 50; the pathogen is Acinetobacterbaumannii and the probe comprises or consists of the nucleic acidsequence set forth as SEQ ID NO: 14; the pathogen is Klebsiellapneumoniae and the probe comprises or consists of the nucleic acidsequence set forth as SEQ ID NO: 20 or SEQ ID NO: 60; the pathogen isToxoplasma gondii and the probe comprises or consists of the nucleicacid sequence set forth as SEQ ID NO: 23; the pathogen is Moraxellacatarrhalis and the probe comprises or consists of the nucleic acidsequence set forth as SEQ ID NO: 26; the pathogen is Escherichia coliand/or Shigella and the probe comprises or consists of the nucleic acidsequence set forth as SEQ ID NO: 29; the pathogen is Staphylococcusaureus and the probe comprises or consists of the nucleic acid sequenceset forth as SEQ ID NO: 32; the pathogen is Chlamydia trachomatis andthe probes comprises or consists of the nucleic acid sequence set forthas SEQ ID NO: 38; the pathogen is Ureaplasma urealyticum and the probecomprises or consists of the nucleic acid sequence set forth as SEQ IDNO: 41; the pathogen is Ureaplasma parvum and the probe comprises orconsists of the nucleic acid sequence set forth as SEQ ID NO: 44; thepathogen is Ureaplasma spp. and the probe comprises or consists of thenucleic acid sequence set forth as SEQ ID NO: 47; and/or the pathogen isStreptococcus agalactiae and the probe comprises or consists of thenucleic acid sequence set forth as SEQ ID NO:
 56. 3-29. (canceled) 30.The method of claim 1, wherein the one or more probes comprise adetectable label.
 31. The method of claim 30, wherein the detectablelabel comprises a donor fluorophore, an acceptor fluorophore, or acombination thereof.
 32. The method of claim 1, further comprisingamplifying a nucleic acid from a pathogen selected from the groupconsisting of Acinetobacter baumannii, Pseudomonas aeruginosa,Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis,Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystisjirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasmaparvum, Ureaplasma spp., Bartonella spp., and a combination of two ormore thereof.
 33. The method of claim 32, wherein the amplifyingcomprises contacting the sample with at least one primer between 10 and40 nucleotides in length comprising a nucleic acid sequence set forth inany one of SEQ ID NOs: 12-13, 15-16, 18-19, 21-22, 24-25, 27-28, 30-31,33-34, 36-37, 39-40, 42-43, 45-46, 48-49, 51, 54-55, and 58-59.
 34. Themethod of claim 33, wherein: the pathogen is Pseudomonas aeruginosa andthe at least one primer comprises the nucleic acid sequence set forth inSEQ ID NO: 15 or SEQ ID NO: 16; the pathogen is Pneumocystis jiroveciiand the at least one primer comprises the nucleic acid sequence setforth in SEQ ID NO: 33 or SEQ ID NO: 34; the pathogen is Bartonella spp.and the at least one primer comprises the nucleic acid sequence setforth in SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 51; the pathogen isAcinetobacter baumannii and the at least one primer comprises thenucleic acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13; thepathogen is Klebsiella pneumoniae and the at least one primer comprisesthe nucleic acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQID NO: 58, or SEQ ID NO: 59; the pathogen is Toxoplasma gondii and theat least one primer comprises the nucleic acid sequence set forth in SEQID NO: 21 or SEQ ID NO: 22; the pathogen is Moraxella catarrhalis andthe at least one primer comprises the nucleic acid sequence set forth inSEQ ID NO: 24 or SEQ ID NO: 25; the pathogen is Escherichia coli and/orShigella and the at least one primer comprises the nucleic acid sequenceset forth in SEQ ID NO: 27 or SEQ ID NO: 28; the pathogen isStaphylococcus aureus and the at least one primer comprises the nucleicacid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31; the pathogenis Chlamydia trachomatis and the at least one primer comprises thenucleic acid sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37; thepathogen is Ureaplasma urealyticum and the at least one primer comprisesthe nucleic acid sequence set forth in SEQ ID NO: 39 or SEQ ID NO: 40;the pathogen is Ureaplasma parvum and the at least one primer comprisesthe nucleic acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 43;the pathogen is Ureaplasma spp. and the at least one primer comprisesthe nucleic acid sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46;and/or the pathogen is Streptococcus agalactiae and the at least oneprimer comprises the nucleic acid sequence set forth in SEQ ID NO: 54 orSEQ ID NO:
 55. 35-47. (canceled)
 48. The method of claim 1, wherein thesample comprises a biological sample or environmental sample.
 49. Themethod of claim 48, wherein the sample is a biological sample comprisingtissue, blood, serum, cerebral spinal fluid, middle ear fluid,bronchoalveolar lavage, tracheal aspirate, sputum, nasopharyngealaspirate, oropharyngeal aspirate, or saliva.
 50. The method of claim 48,wherein the sample is an environmental sample comprising a food sample,a water sample, or a surface swab.
 51. An isolated nucleic acid probe 20to 40 nucleotides in length comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 14, 17, 20, 23, 26, 29, 32, 35,38, 41, 44, 47, 50, 56, and
 60. 52. The isolated nucleic acid probe orclaim 51, wherein the nucleic acid sequence of the probe consists of SEQID NO: 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 56, or 60.53. The isolated nucleic acid probe of claim 51, further comprising adetectable label.
 54. An isolated nucleic acid primer 10 to 40nucleotides in length comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 12, 13, 15, 16, 18, 19, 21, 22, 24,25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51,54, 55, 58, and
 59. 55. The isolated nucleic acid primer of claim 54,wherein the primer consists of a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 12, 13, 15, 16, 18, 19, 21, 22, 24, 25,27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 54,55, 58, and
 59. 56. A kit for detection of a pathogen selected from thegroup consisting of Pseudomonas aeruginosa, Pneumocystis jirovecii,Bartonella spp., Acinetobacter baumannii, Klebsiella pneumoniae,Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella,Staphylococcus aureus, Chlamydia trachomatis, Ureaplasma urealyticum,Ureaplasma parvum, Ureaplasma spp., Streptococcus agalactiae, and acombination of two or more thereof, comprising one or more isolatednucleic acid probes of claim
 51. 57. The kit of claim 56, furthercomprising one or more primers for amplification of a nucleic acid froma pathogen selected from the group consisting of Pseudomonas aeruginosa,Pneumocystis jirovecii, Bartonella spp., Acinetobacter baumannii,Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis,Escherichia coli, Shigella, Staphylococcus aureus, Chlamydiatrachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp.,Streptococcus agalactiae, and a combination of two or more thereof. 58.The kit of claim 57, wherein the one or more primers are selected fromthe group consisting of SEQ ID NOs: 12, 13, 15, 16, 18, 19, 21, 22, 24,25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51,54, 55, 58, and 59.